Method for producing dimerized polypeptide fusions

ABSTRACT

Methods for producing secreted receptor analogs and biologically active peptide dimers are disclosed. The methods for producing secreted receptor analogs and biologically active peptide dimers utilize a DNA sequence encoding a receptor analog or a peptide requiring dimerization for biological activity joined to a dimerizing protein. The receptor analog includes a ligand-binding domain. Polypeptides comprising essentially the extracellular domain of a human PDGF receptor fused to dimerizing proteins, the portion being capable of binding human PDGF or an isoform thereof, are also disclosed. The polypeptides may be used within methods for determining the presence of and for purifying human PDGF or isoforms thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 07/347,291, filed May 2, 1989, which is a continuation-in-partapplication of U.S. application Ser. No. 146,877, filed Jan. 22, 1988,now abandoned.

TECHNICAL FIELD

[0002] The present invention is generally directed toward the expressionof proteins, and more specifically, toward the expression of growthfactor receptor analogs and biologically active dimerized polypeptidefusions.

BACKGROUND OF THE INVENTION

[0003] In higher eucaryotic cells, the interaction between receptors andligands (e.g., hormones) is of central importance in the transmission ofand response to a variety of extracellular signals. It is generallyaccepted that hormones and growth factors elicit their biologicalfunctions by binding to specific recognition sites (receptors) in theplasma membranes of their target cells. Upon ligand binding, a receptorundergoes a conformational change, triggering secondary cellularresponses that result in the activation or inhibition of intracellularprocesses. The stimulation or blockade of such an interaction bypharmacological means has important therapeutic implications for a widevariety of illnesses.

[0004] Ligands fall into two classes: those that have stimulatoryactivity, termed agonists; and those that block the effects elicited bythe original ligands, termed antagonists. The discovery of agonists thatdiffer in structure and composition from the original ligand may bemedically useful. In particular, agonists that are smaller than theoriginal ligand may be especially useful. The bioavailability of thesesmaller agonists may be greater than that of the original ligand. Thismay be of particular importance for topical applications and forinstances when diffusion of the agonist to its target sites is inhibitedby poor circulation. Agonists may also have slightly different spectraof biological activity and/or different potencies, allowing them to beused in very specific situations. Agonists that are smaller andchemically simpler than the native ligand may be produced in greaterquantity and at lower cost. The identification of antagonists whichspecifically block, for example, growth factor receptors has importantpharmaceutical applications. Antagonists that block receptors againstthe action of endogenous, native ligand may be used as therapeuticagents for conditions including atherosclerosis, autocrine tumors,fibroplasia and keloid formation.

[0005] The discovery of new ligands that may be used in pharmaceuticalapplications has centered around designing compounds by chemicalmodification, complete synthesis, and screening potential ligands bycomplex and costly screening procedures. The process of designing a newligand usually begins with the alteration of the structure of theoriginal effector molecule. If the original effector molecule is knownto be chemically simple, for example, a catecholamine or prostaglandin,the task is relatively straightforward. However, if the ligand isstructurally complex, for example, a peptide hormone or a growth factor,finding a molecule which is functionally equivalent to the originalligand becomes extremely difficult.

[0006] Currently, potential ligands are screened using radioligandbinding methods (Lefkowitz et al., Biochem. Biophys. Res. Comm. 60:703-709, 1974; Aurbach et al., Science 186: 1223-1225, 1974; Atlas etal., Proc. Natl. Acad. Sci. USA 71: 4246-4248, 1974). Potential ligandscan be directly assayed by binding the radiolabeled compounds toresponsive cells, to the membrane fractions of disrupted cells, or tosolubilized receptors. Alternatively, potential ligands may be screenedby their ability to compete with a known labeled ligand for cell surfacereceptors.

[0007] The success of these procedures depends on the availability ofreproducibly high quality preparations of membrane fractions or receptormolecules, as well as the isolation of responsive cell lines. Thepreparation of membrane fractions and soluble receptor moleculesinvolves extensive manipulations and complex purification steps. Theisolation of membrane fractions requires gentle manipulation of thepreparation, a procedure which does not lend itself to commercialproduction. It is very difficult to maintain high biological activityand biochemical purity of receptors when they are purified by classicalprotein chemistry methods. Receptors, being integral membrane proteins,require cumbersome purification procedures, which include the use ofdetergents and other solvents that interfere with their biologicalactivity. The use of these membrane preparations in ligand bindingassays typically results in low reproducibility due to the variabilityof the membrane preparations.

[0008] As noted above, ligand binding assays require the isolation ofresponsive cell lines. Often, only a limited subset of cells isresponsive to a particular agent, and such cells may be responsive onlyunder certain conditions. In addition, these cells may be difficult togrow in culture or may possess a low number of receptors. Currentlyavailable cell types responsive to platelet-derived growth factor(PDGF), for example, contain only a low number (up to 4×10⁵; seeBowen-Pope and Ross, J Biol. Chem. 257: 5161-5171, 1982) of receptorsper cell, thus requiring large numbers of cells to assay PDGF analogs orantagonists.

[0009] Presently, only a few naturally-occurring secreted receptors, forexample, the interleukin-2 receptor (IL-2-R) have been identified. Rubinet al. (J. Immun. 135: 3172-3177, 1985) have reported the release oflarge quantities of IL-2-R into the culture medium of activated T-celllines. Bailon et al. (Bio/Technology 5: 1195-1198, 1987) have reportedthe use of a matrix-bound interleukin-2 receptor to purify recombinantinterleukin-2.

[0010] Three other receptors have been secreted from mammalian cells.The insulin receptor (Ellis et al., J Cell Biol. 150: 14a, 1987), theHIV-1 envelope glyco-protein cellular receptor CD4 (Smith et al.,Science 238: 1704-1707, 1987), the murine IL-7 receptor (Cell 60:941-951, 1990) and the epidermal growth factor (EGF) receptor (Livneh etal., J. Biol. Chem. 261: 12490-12497, 1986) have been secreted frommammalian cells using truncated cDNAs that encode portions of theextracellular domains.

[0011] Naturally-occurring, secreted receptors have not been widelyidentified, and there have been only a limited number of reports ofsecreted recombinant receptors. Secreted receptors may be used in avariety of assays, which include assays to determine the presence ofligand in biological fluids and assays to screen for potential agonistsand antagonists. Current methods for ligand screening andligand/receptor binding assays have been limited to those usingpreparations of whole cells or cell membranes for as a source forreceptor molecules. The low reproducibility and high cost of producingsuch preparations does not lend itself to commercial production. Thereis therefore a need in the art for a method of producing secretedreceptors. There is a further need in the art for an assay system thatpermits high volume screening of compounds that may act on highereucaryotic cells via specific surface receptors. This assay systemshould be rapid, inexpensive and adaptable to high volume screening. Thepresent invention discloses such a method and assay system, and furtherprovides other related advantages.

DISCLOSURE OF INVENTION

[0012] Briefly stated, the present invention discloses methods forproducing secreted receptor analogs, including receptor analogs andsecreted platelet-derived growth factor receptor (PDGF-R) analogs. Inaddition, the present invention discloses methods for producing secretedbiologically active dimerized polypeptide fusions.

[0013] Within one aspect of the invention a method for producing asecreted PDGF-R analog is disclosed, comprising (a) introducing into aeukaryotic host cell a DNA construct comprising a transcriptionalpromoter operatively linked to a secretory signal sequence followeddownstream of and in proper reading frame with a DNA sequence encodingat least a portion of the ligand-binding domain of a PDGF-R, the portionincluding a ligand-binding domain; (b) growing the host cell in anappropriate growth medium under physiological conditions to allow thesecretion of a PDGF-R analog encoded by said DNA sequence; and (c)isolating the PDGF-R analog from the host cell.

[0014] Within one embodiment of the present invention, a PDGF-R analogcomprising the amino acid sequence of FIG. 1 (Sequence ID Numbers 1 and2) from isoleucine, number 29, to methionine, number 441, is secreted.Within another embodiment, a PDGF-R analog comprising the amino acidsequence of FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number29 to lysine, number 531 is secreted. Within yet another embodiment ofthe invention, a PDGF-R analog comprising the amino acid sequence ofFIG. 11 (Sequence ID Numbers 35 and 36) from glutamine, number 24 toglutamic acid, number 524 is secreted.

[0015] Yet another aspect of the present invention discloses a methodfor producing a secreted, biologically active dimerized polypeptidefusion. The method generally comprises a) introducing into a eukaryotichost cell a DNA construct comprising a transcriptional promoteroperatively linked to a secretory signal sequence followed downstream byand in proper reading frame with a DNA sequence encoding anon-immunoglobulin polypeptide requiring dimerization for biologicalactivity joined to a dimerizing protein; (b) growing the host cell in anappropriate growth medium under physiological conditions to allow thesecretion of a dimerized polypeptide fusion encodes by said DNAsequence; and (c) isolating the biologically active dimerizedpolypeptide fusion from the host cell.

[0016] Within one embodiment, the dimerizing protein is yeast invertase.Within another embodiment, the dimerizing protein is at least a portionof an immunoglobulin light chain. Within another embodiment, thedimerizing protein is at least a portion of an immunoglobulin heavychain.

[0017] In another aspect of the invention, a method is disclosed forproducing a secreted, biologically active dimerized polypeptide fusion,comprising (a) introducing into a eukaryotic host cell a first DNAconstruct comprising a transcriptional promoter operatively linked to afirst secretory signal sequence followed downstream by and in properreading frame with a first DNA sequence encoding a non-immunoglobulinpolypeptide requiring dimerization for biological activity joined to animmunoglobulin light chain constant region; (b) introducing into thehost cell a second DNA construct comprising a transcriptional promoteroperatively linked to a second secretory signal sequence followeddownstream by and in proper reading frame with a second DNA sequenceencoding an immunoglobulin heavy chain constant region domain selectedfrom the group consisting of C_(H)1, C_(H)2, C_(H)3, and C_(H)4; (c)growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a dimerizedpolypeptide fusion encoded by said first and second DNA sequences; and(d) isolating the dimerized polypeptide fusion from the host cell. Inone embodiment, the second DNA sequence further encodes animmunoglobulin heavy chain hinge region wherein the hinge region isjoined to the heavy chain constant region domain. In a preferredembodiment, the second DNA sequence further encodes an immunoglobulinvariable region joined upstream of and in proper reading frame with theimmunoglobulin heavy chain constant region.

[0018] In another aspect of the invention, a method is disclosed forproducing a secreted, biologically active dimerized polypeptide fusion,comprising (a) introducing into a eukaryotic host cell a first DNAconstruct comprising a transcriptional promoter operatively linked to afirst secretory signal sequence followed downstream by and in properreading frame with a first DNA sequence encoding a non-immunoglobulinpolypeptide requiring dimerization for biological activity joined to animmunoglobulin heavy chain constant region domain selected from thegroup consisting of C_(H)1, C_(H)2, C_(H)3, and C_(H)4; (b) introducinginto the host cell a second DNA construct comprising a transcriptionalpromoter operatively linked to a second secretory signal sequencefollowed downstream by and in proper reading frame with a second DNAsequence encoding an immunoglobulin light chain constant region; (c)growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a dimerizedpolypeptide fusion encoded by said first and second DNA sequences; and(d) isolating the dimerized polypeptide fusion from the host cell. Inone embodiment, the first DNA sequence further encodes an immunoglobulinheavy chain hinge region wherein the hinge region is joined to the heavychain constant region domain. In a preferred embodiment, the second DNAsequence further encodes an immunoglobulin variable region joinedupstream of and in proper reading frame with the immunoglobulin lightchain constant region.

[0019] In another aspect of the invention, a method is disclosed forproducing a secreted, biologically active dimerized polypeptide fusion,comprising (a) introducing into a eukaryotic host cell a DNA constructcomprising a transcriptional promoter operatively linked to a secretorysignal sequence followed downstream by and in proper reading frame witha DNA sequence encoding a nonimmunoglobulin polypeptide requiringdimerization for biological activity joined to an immunoglobulin heavychain constant region domain selected from the group consisting ofC_(H)1, C_(H)2, C_(H)3, and CH₄; (b) growing the host cell in anappropriate growth medium under physiological conditions to allow thesecretion of a dimerized polypeptide fusion encoded by said first andsecond DNA sequences; and (c) isolating the biologically activedimerized polypeptide fusion from the host cell. In one embodiment, theDNA sequence further encodes an immunoglobulin heavy chain hinge regionwherein the hinge region is joined to the heavy chain constant regiondomain.

[0020] In another aspect of the invention, a method is disclosed forproducing a secreted, biologically active dimerized polypeptide fusion,comprising (a) introducing into a eukaryotic host cell a first DNAconstruct comprising a transcriptional promoter operatively linked to afirst secretory signal sequence followed downstream by and in properreading frame with a first DNA sequence encoding a first polypeptidechain of a non-immunoglobulin polypeptide dimer requiring dimerizationfor biological activity joined to an immunoglobulin heavy chain constantregion domain, selected from the group consisting of C_(H)1, C_(H)2,C_(H)3, and C_(H)4; (b) introducing into the host cell a second DNAconstruct comprising a transcriptional promoter operatively linked to asecond secretory signal sequence followed downstream by and in properreading frame with a second DNA sequence encoding a second polypeptidechain of the non-immunoglobulin polypeptide dimer joined to animmunoglobulin light chain constant region domain; (c) growing the hostcell in an appropriate growth medium under physiological conditions toallow the secretion of a dimerized polypeptide fusion encoded by saidfirst and second DNA sequences wherein said dimerized polypeptide fusionexhibits biological activity characteristic of said non-immunoglobulinpolypeptide dimer; and (d) isolating the dimerized polypeptide fusionfrom the host cell. In one embodiment the first DNA sequence furtherencodes an immunoglobulin heavy chain hinge region domain wherein thehinge region is joined to the immunoglobulin heavy chain constant regiondomain.

[0021] Within one embodiment of the present invention, a biologicallyactive dimerized polypeptide fusion comprising the amino acid sequenceof FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number 29, tomethionine, number 441, is secreted. Within another embodiment, abiologically active dimerized polypeptide fusion comprising the aminoacid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine,number 29 to lysine, number 531 is secreted. Within another embodimentof the invention, a biologically active dimerized polypeptide fusioncomprising the amino acid sequence of FIG. 11 (Sequence ID Numbers 35and 36) from glutamine, number 24 to glutamic acid, number 524 issecreted. Within yet another embodiment of the invention, a biologicallyactive dimerized polypeptide fusion comprising the amino acid sequenceof FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number 29 tolysine, number 531 dimerized to the amino acid sequence of FIG. 11(Sequence ID Numbers 35 and 36) from glutamine, number 24 to glutamicacid, number 524 is secreted.

[0022] In yet another aspect of the invention, a method is disclosed forproducing a secreted receptor analog, comprising (a) introducing into aeukaryotic host cell a DNA construct comprising a transcriptionalpromoter operatively linked to at least one secretory signal sequencefollowed downstream by and in proper reading frame with a DNA sequenceencoding a ligand-binding domain of a receptor requiring dimerizationfor biological activity joined to a dimerizing protein; (b) growing thehost cell in an appropriate growth medium under physiological conditionsto allow the secretion of a receptor analog encoded by said DNAsequence; and (c) isolating the receptor analog from the host cell.

[0023] In yet another aspect of the invention, a method is disclosed forproducing a secreted receptor analog, comprising (a) introducing into aeukaryotic host cell a first DNA construct comprising a transcriptionalpromoter operatively linked to a first secretory signal sequencefollowed downstream by and in proper reading frame with a first DNAsequence encoding a ligand-binding domain of a receptor requiringdimerization for biological activity joined to an immunoglobulin lightchain constant region; (b) introducing into the host cell a second DNAconstruct comprising a transcriptional promoter operatively linked to asecond secretory signal sequence followed downstream by and in properreading frame with a second DNA sequence encoding an immunoglobulinheavy chain constant region domain, selected from the group consistingof C_(H)1, C_(H)2, C_(H)3, and C_(H)4; (c) growing the host cell in anappropriate growth medium under physiological conditions to allow thesecretion of a receptor analog encoded by said first and second DNAsequences; and (d) isolating the receptor analog from the host cell. Inone embodiment, the second DNA sequence further encodes animmunoglobulin heavy chain hinge region wherein the hinge region isjoined to the heavy chain constant region domain. In a preferredembodiment, the second DNA sequence further encodes an immunoglobulinvariable region joined upstream of and in proper reading frame with theimmunoglobulin heavy chain constant region.

[0024] In another aspect of the invention, a method is disclosed forproducing a secreted receptor analog, comprising (a) introducing into aeukaryotic host cell a DNA construct comprising a transcriptionalpromoter operatively linked to a secretory signal sequence followeddownstream by and in proper reading frame with a DNA sequence encoding aligand-binding domain of a receptor requiring dimerization forbiological activity joined to an immunoglobulin heavy chain constantregion domain, selected from the group C_(H)1, C_(H)2, C_(H)3, andC_(H)4; (b) growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of the receptor analog;and (c) isolating the receptor analog from the host cell. In oneembodiment, the DNA sequence further encodes an immunoglobulin heavychain hinge region wherein the hinge region is joined to the heavy chainconstant region domain.

[0025] In another aspect of the invention, a method is disclosed forproducing a secreted receptor analog, comprising (a) introducing into aeukaryotic host cell a first DNA construct comprising a transcriptionalpromoter operatively linked to a first secretory signal sequencefollowed downstream of and in proper reading frame with a first DNAsequence encoding a ligand-binding domain of a receptor requiringdimerization for biological activity joined to an immunoglobulin heavychain constant region domain, selected from the group C_(H)1, C_(H)2,C_(H)3, and C_(H)4; (b) introducing into the host cell a second DNAconstruct comprising a transcriptional promoter operatively linked to asecond secretory signal sequence followed downstream by and in properreading frame with a second DNA sequence encoding an immunoglobulinlight chain constant region; (c) growing the host cell in an appropriategrowth medium under physiological conditions to allow the secretion of areceptor analog encoded by said first and second DNA sequences; and (d)isolating the receptor analog from the host cell. In one embodiment, thefirst DNA sequence further encodes an immunoglobulin heavy chain hingeregion wherein the hinge region is joined to the heavy chain constantregion domain. In a preferred embodiment, the second DNA sequencefurther encodes an immunoglobulin variable region joined upstream of andin proper reading frame with the immunoglobulin light chain constantregion.

[0026] In another aspect of the invention, a method is disclosed forproducing a secreted receptor analog, comprising (a) introducing into aeukaryotic host cell a first DNA construct comprising a transcriptionalpromoter operatively linked to a first secretory signal sequencefollowed downstream in proper reading frame by a first DNA sequenceencoding a first polypeptide chain of a ligand-binding domain of areceptor requiring dimerization for biological activity joined to animmunoglobulin heavy chain constant region domain, selected from thegroup C_(H)1, C_(H)2, C_(H)3, and C_(H)4; (b) introducing into the hostcell a second DNA construct comprising a transcriptional promoteroperatively linked to a second secretory signal sequence followeddownstream by and in proper reading frame with a second DNA sequenceencoding a second polypeptide chain of the ligand-binding domain of saidreceptor joined to an immunoglobulin light chain constant region domain;(c) growing the host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a receptor analogencoded by said first and second DNA sequences; and (d) isolating thereceptor analog from the host cell. In one embodiment the first DNAsequence further encodes an immunoglobulin heavy chain hinge regiondomain wherein the hinge region is joined to the immunoglobulin heavychain constant region domain.

[0027] Host cells for use in the present invention include culturedmammalian cells and fungal cells. In a preferred embodiment strains ofthe yeast Saccharomyces cerevisiae are used as host cells. Withinanother preferred embodiment cultured rodent myeloma cells are used ashost cells.

[0028] Within one embodiment of the present invention, a receptor analogis a PDGF-R analog comprising the amino acid sequence of FIG. 1(Sequence ID Numbers 1 and 2) from isoleucine, number 29, to methionine,number 441. Within another embodiment a PDGF-R analog comprises theamino acid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) fromisoleucine, number 29, to lysine, number 531. Within another embodimentof the invention, a PDGF-R analog comprises the amino acid sequence ofFIG. 11 (Sequence ID Numbers 35 and 36) from glutamine, number 24 toglutamic acid, number 524. is secreted. Within yet another embodiment ofthe invention, a PDGF-R analog comprises the amino acid sequence of FIG.1 (Sequence ID Numbers 1 and 2) from isoleucine, number 29 to lysine,number 531 and the amino acid sequence of FIG. 11 (Sequence ID Numbers35 and 36) from glutamine, number 24 to glutamic acid, number 524 issecreted.

[0029] PDGF-R analogs produced by the above-disclosed methods may beused, for instance, within a method for determining the presence ofhuman PDGF or an isoform thereof in a biological sample.

[0030] A method for determining the presence of human PDGF or an isoformthereof in a biological sample is disclosed and comprises (a) incubatinga polypeptide comprising a PDGF receptor analog fused to a dimerizingprotein with a biological sample suspected of containing PDGF or anisoform thereof under conditions that allow the formation ofreceptor/ligand complexes; and (b) detecting the presence ofreceptor/ligand complexes, and therefrom determining the presence ofPDGF or an isoform thereof. Suitable biological samples in this regardinclude blood, urine, plasma, serum, platelet and other cell lysates,platelet releasates, cell suspensions, cell-conditioned culture media,and chemically or physically separated portions thereof.

[0031] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andattached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032]FIG. 1 (Sequence ID Numbers 1 and 2) illustrates the nucleotidesequence of a representative PDGF β-receptor cDNA and the derived aminoacid sequence of the primary translation product and corresponds toSequence ID Number 1). Numbers above the lines refer to the nucleotidesequence; numbers below the lines refer to the amino acid sequence.

[0033]FIG. 2 illustrates the construction of pBTL10, pBTL11 and pBTL12.

[0034]FIG. 3 illustrates the construction of pCBS22.

[0035]FIG. 4 illustrates the construction of pBTL13 and pBTL14.

[0036]FIG. 5 illustrates the construction of pBTL15.

[0037]FIG. 6 illustrates the construction of pBTL22 and pBTL26.

[0038]FIG. 7 illustrates the construction of pSDL114. Symbols used areS.S., signal sequence, C_(k), immunoglobulin light chain constant regionsequence; μ prom, μ promoter, μ enh; μ enhancer.

[0039]FIG. 8 illustrates the construction of pSDLB113. Symbols used areS.S., signal sequence; C_(H)1, C_(H)2, C_(H)3, immunoglobulin heavychain constant region domain sequences; H, immunoglobulin heavy chainhinge region sequence; M, immunoglobulin membrane anchor sequences;C_(γ)1M, immunoglobulin heavy chain constant region and membrane anchorsequences.

[0040]FIG. 9 illustrates the constructions pBTL115, pBTL114,pφ5V_(H)HuC_(γ)1M-neo, plCφ5V_(κ)HuC_(κ)-neo. Symbols used are set forthin FIGS. 7 and 8, and also include L_(H), mouse immunoglobulin heavychain signal sequence; V_(H), mouse immunoglobulin heavy chain variableregion sequence; E, mouse immunoglobulin heavy chain enhancer sequence;L_(κ), mouse immunoglobulin light chain signal sequence; φ5V_(κ), mouseimmunoglobulin light chain variable region sequence; Neo^(R) , E colineomycin resistance gene.

[0041]FIG. 10 illustrates the constructions Zem229R, pφ5V_(H)Fab-neo andpWKI. Symbols used are set forth in FIG. 9.

[0042]FIG. 11 illustrates the sequence of a representative PDGFα-receptor cDNA and the deduced amino acid sequence (using standardone-letter codes) encoded by the cDNA and corresponds to Sequence IDNumbers 35 and 36. Numbers at the ends of the lines refer to nucleotidepositions. Numbers below the sequence refer to amino acid positions.

[0043]FIG. 12 illustrates the assembly of a cDNA molecule encoding aPDGF α-receptor. Complementary DNA sequences are shown as lines. Onlythose portions of the vectors adjacent to the cDNA inserts are shown.

DETAILED DESCRIPTION OF THE INVENTION

[0044] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter.

[0045] DNA Construct: A DNA molecule, or a clone of such a molecule,either single- or double-stranded that has been modified through humanintervention to contain segments of DNA combined and juxtaposed in amanner that as a whole would not otherwise exist in nature.

[0046] DNA constructs contain the information necessary to direct theexpression and/or secretion of DNA sequences encoding polypeptides ofinterest. DNA constructs will generally include promoters, enhancers andtranscription terminators. DNA constructs containing the informationnecessary to direct the secretion of a polypeptide will also contain atleast one secretory signal sequence.

[0047] Secretory Signal Sequence: A DNA sequence encoding a secretorypeptide. A secretory peptide is an amino acid sequence that acts todirect the secretion of a mature polypeptide or protein from a cell.Secretory peptides are characterized by a core of hydrophobic aminoacids and are typically (but not exclusively) found at the amino terminiof newly sythesized proteins. Very often the secretory peptide iscleaved from the mature protein during secretion. Such secretorypeptides contain processing sites that allow cleavage of the signalpeptides from the mature proteins as it passes through the secretorypathway. Processing sites may be encoded within the signal peptide ormay be added to the signal peptide by, for example, in vitromutagenesis. Certain secretory peptides may be used in concert to directthe secretion of polypeptides and proteins. One such secretory peptidethat may be used in combination with other secretory peptides is thethird domain of the yeast Barrier protein.

[0048] Receptor Analog: A non-immunoglobulin polypeptide comprising aportion of a receptor which is capable of binding ligand and/or arerecognized by anti-receptor antibodies. The amino acid sequence of thereceptor analog may contain additions, substitutions or deletions ascompared to the native receptor sequence. A receptor analog may be, forexample, the ligand-binding domain of a receptor joined to anotherprotein. Platelet-derived growth factor receptor (PDGF-R) analogs may,for example, comprise a portion of a PDGF receptor capable of bindinganti-PDGF receptor antibodies, PDGF, PDGF isoforms, PDGF analogs, orPDGF antagonists.

[0049] Dimerizing Protein: A polypeptide chain having affinity for asecond polypeptide chain, such that the two chains associate underphysiological conditions to form a dimer. The second polypeptide chainmay be the same or a different chain.

[0050] Biological activity: A function or set of activities performed bya molecule in a biological context (i.e., in an organism or an in vitrofacsimile thereof). Biological activities may include the induction ofextracellular matrix secretion from responsive cell lines, the inductionof hormone secretion, the induction of chemotaxis, the induction ofmitogenesis, the induction of differentiation, or the inhibition of celldivision of responsive cells. A recombinant protein or peptide isconsidered to be biologically active if it exhibits one or morebiological activities of its native counterpart.

[0051] Ligand: A molecule capable of being bound by the ligand-bindingdomain of a receptor or by a receptor analog. The molecule may bechemically synthesized or may occur in nature. Ligands may be groupedinto agonists and antagonists. Agonists are those molecules whosebinding to a receptor induces the response pathway within a cell.Antagonists are those molecules whose binding to a receptor blocks theresponse pathway within a cell.

[0052] Joined: Two or more DNA coding sequences are said to be joinedwhen, as a result of in-frame fusions between the DNA coding sequencesor as a result of the removal of intervening sequences by normalcellular processing, the DNA coding sequences are translated into apolypeptide fusion.

[0053] As noted above, the present invention provides methods forproducing biologically active dimerized polypeptide fusions and secretedreceptor analogs, which include, for example, PDGF receptor analogs.Secreted receptor analogs may be used to screen for new compounds thatact as agonists or antagonists when interacting with cells containingmembrane-bound receptors. In addition, the methods of the presentinvention provide dimerized non-immunoglobulin polypeptide fusions oftherapeutic value that are biologically active only as dimers. Moreover,the present invention provides methods of producing polypeptide dimersthat are biologically active only as non-covalently associated dimers.Secreted, biologically active dimers that may be produced using thepresent invention include nerve growth factor, colony stimulatingfactor-1, factor XIII, and transforming growth factor β.

[0054] As used herein, the ligand-binding domain of a receptor is thatportion of the receptor that is involved with binding the naturalligand. While not wishing to be bound by theory, the binding of anatural ligand to a receptor is believed to induce a conformationalchange which elicits a response to the change within the responsepathway of the cell. For membrane-bound receptors, the ligand-bindingdomain is generally believed to comprise the extracellular domain forthe receptor. The structure of receptors may be predicted from theprimary translation products using the hydrophobicity plot function of,for example, P/C Gene or Intelligenetics Suite (Intelligenetics, Mt.View, Calif.) or may be predicted according to the methods described,for example, by Kyte and Doolittle, J. Mol. Biol. 157:105-132, 1982).The ligand-binding domain of the PDGF β-receptor, for example, has beenpredicted to include amino acids 29-531 of the published sequence(Gronwald et al., ibid.). The ligand-binding domain of the PDGFα-receptor has been predicted to include amino acids 25-500 of thepublished α-receptor sequence (Matsui et al., ibid.). As used herein,the ligand-binding domain of the PDGF β-receptor includes amino acids29-441 of the sequence of FIG. 1 (Sequence ID Number 1) and C-terminalextensions up to and including amino acid 531. The ligand-binding domainof the PDGF α-receptor is understood to include amino acids 24-524 ofFIG. 11 (Sequence ID Numbers 35 and 36).

[0055] Receptor analogs that may be used in the present inventioninclude the ligand-binding domains of the epidermal growth factorreceptor (EGF-R) and the insulin receptor. As used herein, aligand-binding domain is that portion of the receptor that is involvedin binding ligand and is generally a portion or essentially all of theextracellular domain that extends from the plasma membrane into theextracellular space. The ligand-binding domain of the EGF-R, forexample, resides in the extracellular domain. EGF-R dimers have beenfound to exhibit higher ligand-binding affinity than EGF-R monomers(Boni-Schnetzler and Pilch, Proc. Natl. Acad. Sci. USA 84:7832-7836,1987). The insulin receptor (Ullrich et al., Nature 313:756-761, 1985)requires dimerization for biological activity.

[0056] Another example of a receptor that may be secreted from a hostcell is a platelet-derived growth factor receptor (PDGF-R). Two classesof PDGF-Rs, which recognized different isoforms of PDGF, have beenidentified. (PDGF is a disulfide-bonded, two-chain molecule, which ismade up of an A chain and a B chain. These chains may be combined as ABheterodimers, AA homodimers or BB homodimers. These dimeric moleculesare referred to herein as “isoforms”.) The β-receptor (PDGFβ-R), whichrecognizes only the BB isoform of PDGF (PDGF-BB), has been described(Claesson-Welsh et al., Mol. Cell. Biol. 8:3476-3486, 1988; Gronwald etal., Proc. Natl. Acad. Sci. USA 85:3435-3439, 1988). The α-receptor(PDGFα-R), which recognizes all three PDGF isoforms (PDGF-AA, PDGF-ABand PDGF-BB), has been described by Matsui et al. (Science 243:800-804,1989) and Kelly and Murray (pending commonly assigned U.S. patentapplication Ser. No. 07/355,018, which is incorporated herein byreference). The primary translation products of these receptors indicatethat each includes an extracellular domain implicated in theligand-binding process, a transmembrane domain, and a cytoplasmic domaincontaining a tyrosine kinase activity.

[0057] The present invention provides a standardized assay system, notpreviously available in the art, for determining the presence of PDGF,PDGF isoforms, PDGF agonists or PDGF antagonists using a secreted PDGFreceptor analogs. Such an assay system will typically involve combiningthe secreted PDGF receptor analog with a biological sample underphysiological conditions which permit the formation of receptor-ligandcomplexes, followed by detecting the presence of the receptor-ligandcomplexes. The term physiological conditions is meant to include thoseconditions found within the host organism and include, for example, theconditions of osmolarity, salinity and pH. Detection may be achievedthrough the use of a label attached to the PDGF receptor analog orthrough the use of a labeled antibody which is reactive with thereceptor analog or the ligand. A wide variety of labels may be utilized,such as radionuclides, fluorophores, enzymes and luminescers.Receptor-ligand complexes may also be detected visually, i.e., inimmunoprecipitation assays which do not require the use of a label. Thisassay system provides secreted PDGF receptor analogs that may beutilized in a variety of screening assays for, for example, screeningfor analogs of PDGF. The present invention also provides a methods formeasuring the level of PDGF and PDGF isoforms in biological fluids.

[0058] As noted above, the present invention provides methods forproducing dimerized polypeptide fusions that require dimerization forbiological activity or enhancement of biological activity. Polypeptidesrequiring dimerization for biological activity include, in addition tocertain receptors, nerve growth factor, colony-stimulating factor-1(CSF-1), transforming growth factor β (TGF-β), PDGF, and factor XIII.Nerve growth factor is a non-covalently linked dimer (Harper et al., J.Biol. Chem. 257: 8541-8548, 1982). CSF-1, which specifically stimulatesthe proliferation and differentiation of cells of mononuclear phagocyticlineage, is a disulfide-bonded homodimer (Retternmier et al., Mol. Cell.Biol. 7: 2378-2387, 1987). TGF-β is biologically active as adisulfide-bonded dimer (Assoian et al., J. Biol. Chem. 258: 7155-7160,1983). Factor XIII is a plasma protein that exists as a two chainhomodimer in its activated form (Ichinose et al., Biochem. 25:6900-6906, 1986). PDGF, as noted above, is a disulfide-bonded, two chainmolecule (Murray et al., U.S. Pat. 4,766,073).

[0059] The present invention provides methods by which receptor analogs,including receptor analogs and PDGF-R analogs, requiring dimerizationfor activity may be secreted from host cells. The methods describedherein are particularly advantageous in that they allow the productionof large quantities of purified receptors. The receptors may be used inassays for the screening of potential ligands, in assays for bindingstudies, as imaging agents, and as agonists and antagonists withintherapeutic agents.

[0060] A DNA sequence encoding a human PDGF receptor may be isolated asa cDNA using techniques known in the art (see, for example, Okayama andBerg, Mol. Cell. Biol. 2: 161-170, 1982; Mol. Cell. Biol. 3; 280-289,1983) from a library of human genomic or cDNA sequences. Such librariesmay be prepared by standard procedures, such as those disclosed byGubler and Hoffman (Gene 263-269, 1983). It is preferred that themolecule is a cDNA molecule because cDNA lack introns and are thereforemore suited to manipulation and expression in transfected or transformedcells. Sources of mRNA for use in the preparation of a cDNA libraryinclude the MG-63 human osteosarcoma cell line (available from ATCCunder accession number CRL 1427), diploid human dermal fibroblasts andhuman embryo fibroblast and brain cells (Matsui et al., ibid.). A cDNAencoding a PDGFβ-R has been cloned from a diploid human dermalfibroblast cDNA library using oligonucleotide probes complementary tosequences of the mouse PDGF receptor (Gronwald et al., ibid.). A PDGFα-RcDNA has been isolated by Matsui et al. (ibid.) from human embryofibroblast and brain cells. Alternatively, a cDNA encoding a PDGFα-R maybe isolated from a library prepared from MG-63 human osteosarcoma cellsusing a cDNA probe containing sequences encoding the transmembrane andcytoplasmic domains of the PDGFβ-R (described by Kelly and Murray,ibid.). Partial cDNA clones (fragments) can be extended by re-screeningof the library with the cloned cDNA fragment until the full sequence isobtained. In one embodiment, a ligand-binding domain of a PDGF receptoris encoded by the sequence of FIG. 1 (Sequence ID Number 1) from aminoacid 29 through amino acid 441. In another embodiment, a ligand-bindingdomain of a PDGF receptor is encoded by the sequence of FIG. 1 (SequenceID Number 1) from amino acid 29 through amino acid 531. In yet anotherembodiment, a ligand-binding domain of a PDGF receptor is encoded by thesequence of FIG. 11 (Sequence ID Numbers 35 and 36) from amino acid 24through amino acid 524. One skilled in the art may envision the use ofsmaller DNA sequence encoding the ligand-binding domain of a PDGFreceptor containing at least 400 amino acids of the extracellulardomain.

[0061] DNA sequences encoding EGF-R (Ullrich et al., Nature 304:418-425, 1984), the insulin receptor (Ullrich et al., Nature 313:756-761, 1985), nerve growth factor (Ullrich et al. Nature 303: 821-825,1983), colony stimulating factor-1 (Rettenmier et al., ibid.),transforming growth factor β (Derynck et al., Nature 316: 701-705,1985), PDGF (Murray et al., ibid.), and factor XIII (Ichinose et al.,ibid.) may also be used within the present invention.

[0062] To direct polypeptides requiring dimerization for biologicalactivity or receptor analogs into the secretory pathway of the hostcell, at least one secretory signal sequence is used in conjunction withthe DNA sequence of interest. Preferred secretory signals include thealpha factor signal sequence (pre-pro sequence) (Kurjan and Herkowitz,Cell 30: 933-943, 1982; Kurjan et al., U.S. Pat. No. 4,546,082; Brake,EP 116,201, 1983), the PHO5 signal sequence (Beck et al., WO 86/00637),the BAR1 secretory signal sequence (MacKay et al., U.S. Pat. No.4,613,572; MacKay, WO 87/002670), immunoglobulin V_(H) signal sequences(Loh et al., Cell 33: 85-93, 1983; Watson Nuc. Acids. Res. 12:5145-5164, 1984) and immunoglobulin V_(κ) signal sequences (Watson,ibid.). Particularly preferred signal sequences are the SUC2 signalsequence (Carlson et al., Mol. Cell. Biol. 3: 439-447, 1983) and PDGFreceptor signal sequences. Alternatively, secretory signal sequences maybe synthesized according to the rules established, for example, by vonHeinje (Eur. J. Biochem. 133: 17-21, 1983; J. Mol. Biol. 184: 99-105,1985; Nuc. Acids. Res. 14: 4683-3690, 1986).

[0063] Secretory signal sequences may be used singly or may be combined.For example, a first secretory signal sequence may be used singly orcombined with a sequence encoding the third domain of Barrier (describedin copending commonly assigned U.S. patent application Ser. No.07/104,316, which is incorporated by reference herein in its entirety).The third domain of Barrier may be positioned in proper reading frame 3′of the DNA sequence of interest or 5′ to the DNA sequence and in properreading frame with both the secretory signal sequence and the DNAsequence of interest.

[0064] In one embodiment of the present invention, a sequence encoding adimerizing protein is joined to a sequence encoding a polypeptide chainof a polypeptide dimer or a receptor analog, and this fused sequence isjoined in proper reading frame to a secretory signal sequence. As shownherein, the present invention utilizes such an arrangement to drive theassociation of the polypeptide or receptor analog to form a biologicallyactive molecule upon secretion. Suitable dimerizing proteins include theS. cerevisiae repressible acid phosphatase (Mizunaga et al., J. Biochem.(Tokyo) 103: 321-326, 1988), the S. cerevisiae type 1 killer preprotoxin(Sturley et al., EMBO J. 5: 3381-3390, 1986), the S. calsberaensis alphagalactosidase melibiase (Sumner-Smith et al., Gene 36: 333-340, 1985),the S. cerevisiae invertase (Carlson et al., Mol. Cell. Biol. 3:439-447, 1983), the Neurospora crassa ornithine decarboxylase (Digangiet al., J. Biol. Chem. 262: 7889-7893, 1987), immunoglobulin heavy chainhinge regions (Takahashi et al., Cell 29: 671-679, 1982), and otherdimerizing immunoglobulin sequences. In a preferred embodiment, S.cerevisiae invertase is used to drive the association of polypeptidesinto dimers. Portions of dimerizing proteins, such as those mentionedabove, may be used as dimerizing proteins where those portions arecapable of associating as a dimer in a covalent or noncovlent manner.Such portions may be determined by, for example, altering a sequenceencoding a dimerizing protein through in vitro mutagenesis to deleteportions of the coding sequence. These deletion mutants may be expressedin the appropriate host to determine which portions retain the capablityof associating as dimers. Portions of immunoglobulin gene sequences maybe used to drive the association of non-immunoglobulin polypeptides.These portions correspond to discrete domains of immunoglobulins.Immunoglobulins comprise variable and constant regions, which in turncomprise discrete domains that show similarity in theirthree-dimensional conformations. These discrete domains correspond toimmunoglobulin heavy chain constant region domain exons, immunoglobulinheavy chain variable region domain exons, immunoglobulin light chainvariable region domain exons and immunoglobulin light chain constantregion domain exons in immunoglobulin genes (Hood et al., in Immunology,The Benjamin/Cummings Publishing Company, Inc., Menlo Park, Calif.;Honjo et al., Cell 18: 559-568, 1979; Takahashi et al., Cell 29:671-679, 1982; and Honjo, Ann. Rev. Immun. 1:499-528, 1983)).Particularly preferred portions of immunoglobulin heavy chains includeFab and Fab′ fragments. (An Fab fragment is a portion of animmunoglobulin heavy chain that includes a heavy chain variable regiondomain and a heavy chain constant region domain. An Fab′ fragment is aportion of an immunoglobulin heavy chain that includes a heavy chainvariable region domain, a heavy chain constant region domain and a heavychain hinge region domain.)

[0065] It is preferred to use an immunoglobulin light chain constantregion in association with at least one immunoglobulin heavy chainconstant region domain. In a another embodiment, an immunoglobulin lightchain constant region is associated with at least one immunoglobulinheavy chain constant region domain joined to an immunoglobulin hingeregion. In one set of embodiments, an immunoglobulin light chainconstant region joined in frame with a polypeptide chain of anon-immunoglobulin polypeptide dimer or receptor analog and isassociated with at least one heavy chain constant region. In a preferredset of embodiments a variable region is joined upstream of and in properreading frame with at least one immunoglobulin heavy chain constantregion. In another set of embodiments, an immunoglobulin heavy chain isjoined in frame with a polypeptide chain of a non-immunoglobulinpolypeptide dimer or receptor analog and is associated with animmunoglobulin light chain constant region. In yet another set ofembodiments, a polypeptide chain of a non-immunoglobulin polypeptidedimer or receptor analog joined is to at least one immunoglobulin heavychain constant region which is joined to an immunoglobulin hinge regionand is associated with an immunoglobulin light chain constant region. Ina preferred set of embodiments an immunoglobulin variable region isjoined upstream of and in proper reading frame with the immunoglobulinlight chain constant region.

[0066] Immunoglobulin heavy chain constant region domains includeC_(H)1, C_(H)2, C_(H)3, and C_(H)4 of any class of immunoglobulin heavychain including γ, α, ε, μ, and δ classes (Honjo, ibid., 1983) Aparticularly preferred immunoglobulin heavy chain constant region domainis human C_(H)1. Immunoglobulin variable regions include V_(H), V_(κ),or V_(λ).

[0067] DNA sequences encoding immunoglobulins may be cloned from avariety of genomic or cDNA libraries known in the art. The techniquesfor isolating such DNA sequences using probe-based methods areconventional techniques and are well known to those skilled in the art.Probes for isolating such DNA sequences may be based on published DNAsequences (see, for example, Hieter et al., Cell 22: 197-207, 1980).Alternatively, the polymerase chain reaction (PCR) method disclosed byMullis et al. (U.S. Pat. No. 4,683,195) and Mullis (U.S. Pat. No.4,683,202), incorporated herein by reference may be used. The choice oflibrary and selection of probes for the isolation of such DNA sequencesis within the level of ordinary skill in the art.

[0068] Host cells for use in practicing the present invention includeeukaryotic cells capable of being transformed or transfected withexogenous DNA and grown in culture, such as cultured mammalian andfungal cells. Fungal cells, including species of yeast (e.g.,Saccharomyces spp., Schizosaccharomyces spp.), or filamentous fungi(e.g., Aspergillus spp., Neurospora spp.) may be used as host cellswithin the present invention. Strains of the yeast Saccharomycescerevisiae are particularly preferred.

[0069] Expression units for use in the present invention will generallycomprise the following elements, operably linked in a 5′ to 3′orientation: a transcriptional promoter, a secretory signal sequence aDNA sequence encoding nonimmunoglobulin polypeptide requiringdimerization for biological activity joined to a dimerizing protein anda transcriptional terminator. The selection of suitable promoters,signal sequences and terminators will be determined by the selected hostcell and will be evident to one skilled in the art and are discussedmore specifically below.

[0070] Suitable yeast vectors for use in the present invention includeYRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978),YEp13 (Broach et al., Gene 8: 121-133, 1979), pJDB249 and pJDB219(Beggs, Nature 275:104-108, 1978) and derivatives thereof. Such vectorswill generally include a selectable marker, which may be one of anynumber of genes that exhibit a dominant phenotype for which a phenotypicassay exists to enable transformants to be selected. Preferredselectable markers are those that complement host cell auxotrophy,provide antibiotic resistance or enable a cell to utilize specificcarbon sources, and include LEU2 (Broach et al. ibid.), URA3 (Botsteinet al., Gene 8: 17, 1979), HIS3 (Struhl et al., ibid.) or POT1 (Kawasakiand Bell, EP 171,142). Other suitable selectable markers include the CATgene, which confers chloramphenicol resistance on yeast cells.

[0071] Preferred promoters for use in yeast include promoters from yeastglycolytic genes (Hitzeman et al., J Biol. Chem. 255: 12073-12080, 1980;Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki,U.S. Pat. No. 4,599,311) or alcohol dehydrogenase genes (Young et al.,in Genetic Engineering of Microorganisms for Chemicals, Hollaender etal., (eds.), p. 355, Plenum, New York, 1982; Ammerer, Meth. Enzymol.101: 192-201, 1983). In this regard, particularly preferred promotersare the TPI1 promoter (Kawasaki, U.S. Pat. No. 4,599,311, 1986) and theADH2-4^(C) promoter (Russell et al., Nature 304: 652-654, 1983 and Iraniand Kilgore, described in pending, commonly assigned U.S. patentapplication Ser. No. 07/183,130, which is incorporated herein byreference). The expression units may also include a transcriptionalterminator. A preferred transcriptional terminator is the TPI1terminator (Alber and Kawasaki, ibid.).

[0072] In addition to yeast, proteins of the present invention can beexpressed in filamentous fungi, for example, strains of the fungiAspergillus (McKnight and Upshall, described in commonly assigned U.S.Pat. No. 4,935,349, which is incorporated herein by reference). Examplesof useful promoters include those derived from Aspergillus nidulansglycolytic genes, such as the ADH3 promoter (McKnight et al., EMBO J. 4:2093-2099, 1985) and the tpiA promoter. An example of a suitableterminator is the ADH3 terminator (McKnight et al., ibid.). Theexpression units utilizing such components are cloned into vectors thatare capable of insertion into the chromosomal DNA of Aspergillus.

[0073] Techniques for transforming fungi are well known in theliterature, and have been described, for instance, by Beggs (ibid.),Hinnen et al. (Proc. Natl. Acad. Sci. USA 75: 1929-1933, 1978), Yeltonet al., (Proc. Natl. Acad. Sci. USA 81: 1740-1747, 1984), and Russell(Nature 301: 167-169, 1983). The genotype of the host cell willgenerally contain a genetic defect that is complemented by theselectable marker present on the expression vector. Choice of aparticular host and selectable marker is well within the level ofordinary skill in the art.

[0074] In a preferred embodiment, a Saccharomyces cerevisiae host cellthat contains a genetic deficiency in a gene required forasparagine-linked glycosylation of glycoproteins is used. Preferably,the S. cerevisiae host cell contains a genetic deficiency in the MNN9gene (described in pending, commonly assigned U.S. patent applicationSer. Nos. 116,095 and 189,547 which are incorporated by reference hereinin their entirety). Most preferably, the S. cerevisiae host cellcontains a disruption of the MNN9 gene. S. cerevisiae host cells havingsuch defects may be prepared using standard techniques of mutation andselection. Ballou et al. (J. Biol. Chem. 255: 5986-5991, 1980) havedescribed the isolation of mannoprotein biosynthesis mutants that aredefective in genes which affect asparagine-linked glycosylation.Briefly, mutagenized S. cerevisiae cells were screened usingfluoresceinated antibodies directed against the outer mannose chainspresent on wild-type yeast. Mutant cells that did not bind antibody werefurther characterized and were found to be defective in the addition ofasparagine-linked oligosaccharide moieties. To optimize production ofthe heterologous proteins, it is preferred that the host strain carriesa mutation, such as the S. cerevisiae pep4 mutation (Jones, Genetics 85:23-33, 1977), which results in reduced proteolytic activity.

[0075] In addition to fungal cells, cultured mammalian cells may be usedas host cells within the present invention. Preferred cell lines arerodent myeloma cell lines, which include p3X63Ag8 (ATCC TIB 9), FO (ATCCCRL 1646), NS-1 (ATCC TIB 18) and 210-RCY-Ag1 (Galfre et al., Nature277: 131, 1979). A particularly preferred rodent myeloma cell line isSP2/0-Ag14 (ATCC CRL 1581). In addition, a number of other cell linesmay be used within the present invention, including COS-1 (ATCC CRL1650), BHK, p363.Ag.8.653 (ATCC CRL 1580) Rat Hep I (ATCC CRL 1600), RatHep II (ATCC CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC CCL 75.1),Human hepatoma (ATCC HTB-52), Hep G2 (ATCC HB 8065), Mouse liver (ATCCCC 29.1), 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72,1977) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci USA 77:4216-4220, 1980) A preferred BHX cell line is the tk⁻ts13 BHK cell line(Waechter and Baserga, Proc. Natl. Acad. Sci USA 79: 1106-1110, 1982). Apreferred BHK cell line is the tk⁻ts13 BHK cell line (Waechter andBaserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982). A tk⁻ BHK cellline is available from the American Type Culture Collection, Rockville,Md. under accession number CRL 1632. A particularly preferred tk⁻ BHKcell line is BHK 570 which is available from the American Type CultureCollection under accession number 10314.

[0076] Mammalian expression vectors for use in carrying out the presentinvention will include a promoter capable of directing the transcriptionof a cloned gene or cDNA. Preferred promoters include viral promotersand cellular promoters. Preferred viral promoters include the major latepromoter from adenovirus 2 (Kaufman and Sharp, Mol Cell. Biol. 2:1304-13199, 1982) and the SV40 promoter (Subramani et al., Mol. Cell.Biol. 1: 854-864, 1981). Preferred cellular promoters include the mousemetallothionein 1 promoter (Palmiter et al., Science 222: 809-814, 1983)and a mouse V_(κ) promoter (Grant et al., Nuc. Acids Res. 15: 5496,1987). A particularly preferred promoter is a mouse V_(H) promoter (Lohet al., ibid.). Such expression vectors may also contain a set of RNAsplice sites located downstream from the promoter and upstream from theDNA sequence encoding the peptide or protein of interest. Preferred RNAsplice sites may be obtained from adenovirus and/or immunoglobulingenes. Also contained in the expression vectors is a polyadenylationsignal located downstream of the coding sequence of interest.Polyadenylation signals include the early or late polyadenylationsignals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signalfrom the adenovirus 5 E1B region and the human growth hormone geneterminator (DeNoto et al., Nuc. Acids Res. 9: 3719-3730, 1981). Aparticularly preferred polyadenylation signal is the V_(H) geneterminator (Loh et al., ibid.). The expression vectors may include anoncoding viral leader sequence, such as the adenovirus 2 tripartiteleader, located between the promoter and the RNA splice sites. Preferredvectors may also include enhancer sequences, such as the SV40 enhancerand the mouse μ enhancer (Gillies, Cell 33: 717-728, 1983). Expressionvectors may also include sequences encoding the adenovirus VA RNAs.

[0077] Cloned DNA sequences may be introduced into cultured mammaliancells by, for example, calcium phosphate-mediated transfection (Wigleret al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1973.) Othertechniques for introducing cloned DNA sequences into mammalian cells,such as electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), mayalso be used. In order to identify cells that have integrated the clonedDNA, a selectable marker is generally introduced into the cells alongwith the gene or cDNA of interest. Preferred selectable markers for usein cultured mammalian cells include genes that confer resistance todrugs, such as neomycin, hygromycin, and methotrexate. The selectablemarker may be an amplifiable selectable marker. A preferred amplifiableselectable marker is the DHFR gene. A particularly preferred amplifiablemarker is the DHFR^(r) cDNA (Simonsen and Levinson, Proc. Natl. Adac.Sci. USA 80: 2495-2499, 1983). Selectable markers are reviewed by Thilly(Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.) andthe choice of selectable markers is well within the level of ordinaryskill in the art.

[0078] Selectable markers may be introduced into the cell on a separateplasmid at the same time as the gene of interest, or they may beintroduced on the same plasmid. If on the same plasmid, the selectablemarker and the gene of interest may be under the control of differentpromoters or the same promoter, the latter arrangement producing adicistronic message. Constructs of this type are known in the art (forexample, Levinson and Simonsen, U.S. Pat. No. 4,713,339). It may also beadvantageous to add additional DNA, known as “carrier DNA” to themixture which is introduced into the cells.

[0079] Transfected mammalian cells are allowed to grow for a period oftime, typically 1-2 days, to begin expressing the DNA sequence(s) ofinterest. Drug selection is then applied to select for growth of cellsthat are expressing the selectable marker in a stable fashion. For cellsthat have been transfected with an amplifiable selectable marker thedrug concentration may be increased in a stepwise manner to select forincreased copy number of the cloned sequences, thereby increasingexpression levels.

[0080] Host cells containing DNA constructs of the present invention aregrown in an appropriate growth medium. As used herein, the term“appropriate growth medium” means a medium containing nutrients requiredfor the growth of cells. Nutrients required for cell growth may includea carbon source, a nitrogen source, essential amino acids, vitamins,minerals and growth factors. The growth medium will generally select forcells containing the DNA construct by, for example, drug selection ordeficiency in an essential nutrient which are complemented by theselectable marker on the DNA construct or co-transfected with the DNAconstruct. Yeast cells, for example, are preferably grown in achemically defined medium, comprising a non-amino acid nitrogen source,inorganic salts, vitamins and essential amino acid supplements. The pHof the medium is preferably maintained at a pH greater than 2 and lessthan 8, preferably at pH 6.5. Methods for maintaining a stable pHinclude buffering and constant pH control, preferably through theaddition of sodium hydroxide. Preferred buffering agents includesuccinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Yeastcells having a defect in a gene required for asparagine-linkedglycosylation are preferably grown in a medium containing an osmoticstabilizer. A preferred osmotic stabilizer is sorbitol supplemented intothe medium at a concentration between 0.1 M and 1.5 M., preferably at0.5 M or 1.0 M. Cultured mammalian cells are generally grown incommercially available serum-containing or serum-free media. Selectionof a medium appropriate for the particular cell line used is within thelevel of ordinary skill in the art.

[0081] The culture medium from appropriately grown transformed ortransfected host cells is separated from the cell material, and thepresence of dimerized polypeptide fusions or secreted receptor analogsis demonstrated. A preferred method of detecting receptor analogs, forexample, is by the binding of the receptors or portions of receptors toa receptor-specific antibody. An anti-receptor antibody may be amonoclonal or polyclonal antibody raised against the receptor inquestion, for example, an anti-PDGF receptor monoclonal antibody may beused to assay for the presence of PDGF-receptor analogs. Anotherantibody, which may be used for detecting substance P-tagged peptidesand proteins, is a commercially available rat anti-substance Pmonoclonal antibody which may be utilized to visualize peptides orproteins that are fused to the C-terminal amino acids of substance P.Ligand binding assays may also be used to detect the presence ofreceptor analogs. In the case of PDGF receptor analogs, it is preferableto use fetal foreskin fibroblasts, which express PDGF receptors, tocompete against the PDGF receptor analogs of the present invention forlabeled PDGF and PDGF isoforms.

[0082] Assays for detection of secreted, biologically active peptidedimers and receptor analogs may include Western transfer, protein blotor colony filter. A Western transfer filter may be prepared using themethod described by Towbin et al. (Proc. Natl. Acad. Sci. USA 76:4350-4354, 1979). Briefly, samples are electrophoresed in a sodiumdodecylsulfate polyacrylamide gel. The proteins in the gel areelectrophoretically transferred to nitrocellulose paper. Protein blotfilters may be prepared by filtering supernatant samples or concentratesthrough nitrocellulose filters using, for example, a Minifold(Schleicher & Schuell, Keene, N.H.). Colony filters may be prepared bygrowing colonies on a nitrocellulose filter that has been laid across anappropriate growth medium. In this method, a solid medium is preferred.The cells are allowed to grow on the filters for at least 12 hours. Thecells are removed from the filters by washing with an appropriate bufferthat does not remove the proteins bound to the filters. A preferredbuffer comprises 25 mM Tris-base, 19 mM glycine, pH 8.3, 20% methanol.

[0083] The dimerized polypeptide fusions and receptor analogs present onthe Western transfer, protein blot or colony filters may be visualizedby specific antibody binding using methods known in the art. Forexample, Towbin et al. (ibid.) describe the visualization of proteinsimmobilized on nitrocellulose filters with a specific antibody followedby a labeled second antibody, directed against the first antibody. Kitsand reagents required for visualization are commercially available, forexample, from Vector Laboratories, (Burlingame, Calif.), and SigmaChemical Company (St. Louis, Mo.)

[0084] Secreted, biologically active dimerized polypeptide fusions andreceptor analogs may be isolated from the medium of host cells grownunder conditions that allow the secretion of the biologically activedimerized polypeptide fusions and receptor analogs. The cell material isremoved from the culture medium, and the biologically active dimerizedpolypeptide fusions and receptor analogs are isolated using isolationtechniques known in the art. Suitable isolation techniques includeprecipitation and fractionation by a variety of chromatographic methods,including gel filtration, ion exchange chromatography and immunoaffinitychromatography. A particularly preferred purification method isimmunoaffinity chromatography using an antibody directed against thereceptor analog or dimerized polypeptide fusion. The antibody ispreferably immobilized or attached to a solid support or substrate. Aparticularly preferred substrate is CNBr-activated Sepharose (PharmaciaLKB Technologies, Inc., Piscataway, N.J.). By this method, the medium iscombined with the antibody/substrate under conditions that will allowbinding to occur. The complex may be washed to remove unbound material,and the receptor analog or peptide dimer is released or eluted throughthe use of conditions unfavorable to complex formation. Particularlyuseful methods of elution include changes in pH, wherein the immobilizedantibody has a high affinity for the ligand at a first pH and a reducedaffinity at a second (higher or lower) pH; changes in concentration ofcertain chaotropic agents; or through the use of detergents.

[0085] The secreted PDGF receptor analogs of the present invention canbe used within a variety of assays for detecting the presence of and/orscreening for native PDGF, PDGF isoforms or PDGF-like molecules. Theseassays will typically involve combining PDGF receptor analogs, which maybe bound to a solid substrate such as polymeric microtiter plate wells,with a biological sample under conditions that permit the formation ofreceptor/ligand complexes. Screening assays for the detection of PDGF,PDGF isoforms or PDGF-like molecules will typically involve combiningsoluble PDGF receptor analogs with a biological sample and incubatingthe mixture with a PDGF isoform or mixture of PDGF isoforms bound to asolid substrate such as polymeric microtiter plates, under conditionsthat permit the formation of receptor/ligand complexes. Detection may beachieved through the use of a label attached to the receptor or throughthe use of a labeled antibody which is reactive with the receptor.Alternatively, the labeled antibody may be reactive with the ligand. Awide variety of labels may be utilized, such as radionuclides,fluorophores, enzymes and luminescers. Complexes may also be detectedvisually, i.e., in immunoprecipitation assays, which do not require theuse of a label.

[0086] Secreted PDGF receptor analogs of the present invention may alsobe labeled with a radioisotope or other imaging agent and used for invivo diagnostic purposes. Preferred radioisotope imaging agents includeiodine-125 and technetium-99, with technetium-99 being particularlypreferred. Methods for producing protein-isotope conjugates are wellknown in the art, and are described by, for example, Eckelman et al.(U.S. Pat. No. 4,652,440), Parker et al. (WO 87/05030) and Wilber et al.(EP 203,764). Alternatively, the secreted receptor analogs may be boundto spin label enhancers and used for magnetic resonance (MR) imaging.Suitable spin label enhancers include stable, sterically hindered, freeradical compounds such as nitroxides. Methods for labeling ligands forMR imaging are disclosed by, for example, Coffman et al. (U.S. Pat. No.4,656,026). For administration, the labeled receptor analogs arecombined with a pharmaceutically acceptable carrier or diluent, such assterile saline or sterile water. Administration is preferably by bolusinjection, preferably intravenously. These imaging agents areparticularly useful in identifying the locations of atheroscleroticplaques, PDGF-producing tumors, and receptor-bound PDGF.

[0087] The secreted PDGF receptor analogs of the present invention mayalso be utilized within diagnostic kits. Briefly, the subject receptoranalogs are preferably provided in a lyophilized form or immobilizedonto the walls of a suitable container, either alone or in conjunctionwith antibodies capable of binding to native PDGF or selected PDGFisoform(s) or specific ligands. The antibodies, which may be conjugatedto a label or unconjugated, are generally included in the kits withsuitable buffers, such as phosphate, stabilizers, inert proteins or thelike. Generally, these materials are present in less than about 5%weight based upon the amount of active receptor analog, and are usuallypresent in an amount of at least about 0.001% weight. It may also bedesirable to include an inert excipient to dilute the activeingredients. Such an excipient may be present from about 1% to 99%weight of the total composition. In addition, the kits will typicallyinclude other standard reagents, instructions and, depending upon thenature of the label involved, reactants that are required to produce adetectable product. Where an antibody capable of binding to the receptoror receptor/ligand complex is employed, this antibody will usually beprovided in a separate vial. The antibody is typically conjugated to alabel and formulated in an analogous manner with the formulationsbriefly described above. The diagnostic kits, including the containers,may be produced and packaged using conventional kit manufacturingprocedures.

[0088] As noted above, the secreted PDGF receptor analogs of the presentinvention may be utilized within methods for purifying PDGF from avariety of samples. Within a preferred method, the secreted PDGFreceptor analogs are immobilized or attached to a substrate or supportmaterial, such as polymeric tubes, beads, polysaccharide particulates,polysaccharide-containing materials, polyacrylamide or other waterinsoluble polymeric materials. Methods for immobilization are well knownin the art (Mosbach et al., U.S. Pat. No. 4,415,665; Clarke et al.,Meth. Enzymology 68: 436-442, 1979). A common method of immobilizationis CNBr activation. Activated substrates are commercially available froma number of suppliers, including Pharmacia (Piscataway, N.J.), PierceChemical Co. (Rockford, Ill.) and Bio-Rad Laboratories (Richmond,Calif.). A preferred substrate is CNBr-activated Sepharose (Pharmacia,Piscataway, N.J.). Generally, the substrate/receptor analog complex willbe in the form of a column. The sample is then combined with theimmobilized receptor analog under conditions that allow binding tooccur. The substrate with immobilized receptor analog is firstequilibrated with a buffer solution of a composition in which thereceptor analog has been previously found to bind its ligand. Thesample, in the solution used for equilibration, is then applied to thesubstrate/receptor analog complex. Where the complex is in the form of acolumn, it is preferred that the sample be passed over the column two ormore times to permit full binding of ligand to receptor analog. Thecomplex is then washed with the same solution to elute unbound material.In addition, a second wash solution may be used to minimize nonspecificbinding. The bound material may then be released or eluted through theuse of conditions unfavorable to complex formation. Particularly usefulmethods include changes in pH, wherein the immobilized receptor has ahigh affinity for PDGF at a first pH and reduced affinity at a second(higher or lower) pH; changes in concentration of certain chaotropicagents; or through the use of detergents.

[0089] The secreted PDGF receptor analogs fused to dimerizing proteinsof the present invention may be used in pharmaceutical compositions fortopical or intravenous application. The secreted PDGF receptor analogsof the present invention may be useful in the treatment ofatherosclerosis by, for example, binding endogenous PDGF to preventsmooth muscle cell proliferation. The PDGF receptor analogs fused todimerizing proteins are used in combination with a physiologicallyacceptable carrier or diluent. Preferred carriers and diluents includesaline and sterile water. Pharmaceutical compositions may also containstabilizers and adjuvants. The resulting aqueous solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile aqueoussolution prior to administration.

[0090] The following examples are offered by way of illustration and notby way of limitation.

EXAMPLES

[0091] Enzymes, including restriction enzymes, DNA polymerase I (Klenowfragment), T4 DNA polymerase, T4 DNA ligase and T4 polynucleotidekinase, were obtained from New England Biolabs (Beverly, Mass.),GIBCO-BRL (Gaithersburg, Md.) and Boerhinger-Mannheim Biochemicals(Indianapolis, Ind.) and were used as directed by the manufacturer or asdescribed in Maniatis et al. (Molecular Cloning: A Laboratorv Manual,Cold Spring Harbor Laboratory, NY, 1982) and Sambrook et al. (MolecularCloning: A Laboratory Manual/Second Edition, Cold Spring HarborLaboratory, NY, 1989).

Example 1 Cloning PDGF Receptor cDNAs

[0092] A. Cloning the PDGF β-Receptor

[0093] A cDNA encoding the PDGF β-receptor was cloned as follows.Complementary DNA (cDNA) libraries were prepared from poly(A) RNA fromdiploid human dermal fibroblast cells, prepared by explant from a normaladult, essentially as described by Hagen et al. (Proc. Natl. Acad. Sci.USA 83: 2412-2416, 1986). Briefly, the poly(A) RNA was primed with oligod(T) and cloned into λgt11 using Eco RI linkers. The random primedlibrary was screened for the presence of human PDGF receptor cDNA'susing three oligonucleotide probes complementary to sequences of themouse PDGF receptor (Yarden et al., Nature 323: 226-232, 1986).Approximately one million phage from the random primed human fibroblastcell library were screened using oligonucleotides ZC904, ZC905 and ZC906(Table 1; Sequence ID Numbers 5, 6 and 7, respectively). Eight positiveclones were identified and plaque purified. Two clones, designated RP41and RP51, were selected for further analysis by restriction enzymemapping and DNA sequence analysis. RP51 was found to contain 356 bp of5′-noncoding sequence, the ATG translation initiation codon and 738 bpof the amino terminal coding sequence. RP41 was found to overlap cloneRP51 and contained 2649 bp encoding amino acids 43-925 of the β-receptorprotein. TABLE 1 Oligonucleotide Sequences ZC871 (Sequence ID Number 3)5′ CTC TCT TCC TCA GGT AAA TGA GTG CCA GGG CCG GCA AGC CCC CGC TCC 3′ZC872 (Sequence ID Number 4) 5′ CCG GGG AGC GGG GGC TTG CCG GCC CTG GCACTC ATT TAC CTG AGG AAG AGA GAG CT 3′ ZC904 (Sequence ID Number 5)5′ CAT GGG CAC GTA ATC TAT AGA TTC ATC CTT GCT CAT ATC CAT GTA 3′ ZC905(Sequence ID Number 6) 5′ TCT TGC CAG GGC ACC TGG GAC ATC TGT TCC CACATC ACC GG 3′ ZC906 (Sequence ID Number 7) 5′ AAG CTG TCC TCT GCT TCAGCC AGA GGT CCT GGG CAG CC 3′ ZC1380 (Sequence ID Number 8) 5′ CAT GGTGGA ATT CCT GCT GAT 3′ ZC1447 (Sequence ID Number 9) 5′ TG GTT GTG CAGAGC TGA GGA AGA GAT GGA 3′ ZC1453 (Sequence ID Number 10) 5′ AAT TCA TTATGT TGT TGC AAG CCT TCT TGT TCC TGC TAG CTG GTT TCG CTG TTA A 3′ ZC1454(Sequence ID Number 11) 5′ GAT CTT AAC AGC GAA ACC AGC TAG CAG GAA CAAGAA GGC TTG CAA CAA CAT AAT G 3′ ZC1478 (Sequence ID Number 12) 5′ ATCGCG AGC ATG CAG ATC TGA 3′ ZC1479 (Sequence ID Number 13) 5′ AGC TTC AGATCT GCA TGC TGC CGA T 3′ ZC1776 (Sequence ID Number 14) 5′ AGC TGA GCGCAA ATG TTG TGT CGA GTG CCC ACC GTG CCC AGC TTA GAA TTC T 3′ ZC1777(Sequence ID Number 15) 5′ CTA GAG AAT TCT AAG CTG GGC ACG GTG GGC ACTCGA CAC AAC ATT TGC GCT C 3′ ZC1846 (Sequence ID Number 16) 5′ GAT CGGCCA CTG TCG GTG CGC TGC ACG CTG CGC AAC GCT GTG GGC CAG GAC ACG CAG GAGGTC ATC GTG GTG CCA CAC TCC TTG CCC TTT AAG CA 3′ ZC1847 (Sequence IDNumber 17) 5′ AGC TTG CTT AAA GGG CAA GGA GTG TGG CAC CAC GAT GAC CTCCTG CGT GTC CTG GCC CAC AGC GTT GCG CAG CGT GCA GCG CAC CGA CAG TGG CC3′ ZC1886 (Sequence ID Number 18) 5′ CCA GTG CCA AGC TTG TCT AGA CTT ACCTTT AAA GGG CAA GGA G 3′ ZC1892 (Sequence ID Number 19) 5′ AGC TTG AGCGT 3′ ZC1893 (Sequence ID Number 20) 5′ CTA GAC GCT CA 3′ ZC1894(Sequence ID Number 21) 5′ AGC TTC CAG TTC TTC GGC CTC ATG TCA GTT CTTCGG CCT CAT GTG AT 3′ ZC1895 (Sequence ID Number 22) 5′ CTA CAT CAC ATGAGG CCG AAG AAC TGA CAT GAG GCC GAA GAA CTG GA 3′ ZC2181 (Sequence IDNumber 23) 5′ AAT TCG GAT CCA CCA TGG GCA CCA GCC ACC CGG CGT TCC TGGTGT TAG GCT GCC TGC TGA CCG GCC 3′ ZC2182 (Sequence ID Number 24) 5′ TGAGCC TGA TCC TGT GCC AAC TGA GCC TGC CAT CGA TCC TGC CAA ACG AGA ACG AGAAGG TTG TGC AGC TA 3′ ZC2183 (Sequence ID Number 25) 5′ AAT TTA GCT GCACAA CCT TCT CGT TCT CGT TTG GCA GGA TCG ATG GCA GGC TCA GTT GGC ACA GGATCA 3′ ZC2184 (Sequence ID Number 26) 5′ GGC TCA GGC CGG TCA GCA GGC AGCCTA ACA CCA GGA ACG CCG GGT GGC TGG TGC CCA TGG TGG ATC CG 3′ ZC2311(Sequence ID Number 27) 5′ TGA TCA CCA TGG CTC AAC TG 3′ ZC2351(Sequence ID Number 28) 5′ CGA ATT CCA C 3′ ZC2352 (Sequence ID Number29) 5′ CAT GGT GGA ATT CGA GCT 3′ ZC2392 (Sequence ID Number 30) 5′ ACGTAA GCT TGT CTA GAC TTA CCT TCA GAA CGC AGG GTG GG 3′

[0094] The 3′-end of the cDNA was not isolated in the first cloning andwas subsequently isolated by screening 6×10⁵ phage of the oligod(T)-primed cDNA library with a 630 bp Sst I-Eco RI fragment derivedfrom the 3′-end of clone RP41. One isolate, designated OT91, was furtheranalyzed by restriction enzyme mapping and DNA sequencing. This clonewas found to comprise the 3′-end of the receptor coding region and 1986bp of 3′ untranslated sequence.

[0095] Clones RP51, RP41 and OT91 were ligated together to construct afull-length cDNA encoding the entire PDGF β-receptor. RP41 was digestedwith Acc I and Bam HI to isolate the 2.12 kb fragment. RP51 was digestedwith Eco RI and Acc I to isolate the 982 bp fragment. The 2.12 kb RP41fragment and the 982 bp RP51 fragment were joined in a three-partligation, with pUC13, which had been linearized by digestion with Eco RIand Bam HI. The resultant plasmid was designated 51/41. Plasmid 51/41was digested with Eco RI and Bam HI to isolate the 3 kb fragmentcomprising the partial PDGF receptor cDNA. OT91 was digested with Bam HIand Xba I to isolate the 1.4 kb fragment containing the 3′ portion ofthe PDGF receptor cDNA. The Eco RI-Bam HI 51/41 fragment, the Bam HI-XbaI OT91 fragment and the Eco RI-Xba I digested pUC13 were joined in athree-part ligation. The resultant plasmid was designated pR-RX1 (FIG.2).

[0096] B. Cloning the PDGF-αReceptor

[0097] A cDNA encoding to PDGF α-receptor was cloned as follows. RNA wasprepared by the method of Chirgwin et al. (Biochemistry 18: 5294, 1979)and twice purified on oligo d(T) cellulose to yield poly(A)+ RNA.Complementary DNA was prepared in λgt10 phage using a kit purchased fromInvitrogen (San Diego, Calif.) The resulting λ phage DNA was packagedwith a coat particle mixture from Stratagene Cloning Systems (La Jolla,Calif.) infected into E. coli strain C600 Hfl⁻ and titered.

[0098] Approximately 1.4×10⁶ phage recombinants were plated to produceplaques for screening. Nitrocellulose filter lifts were preparedaccording to standard methods and were hybridized to a radiolabeled PDGFβ-receptor DNA fragment (Gronwald et al., ibid.) comprising the 1.9 kbFsp I-Hind III fragment that encodes the transmembrane and cytoplasmicdomains of the PDGF β-receptor cDNA. Hybridization was performed for 36hours at 42° C. in a mixture containing 40% formamide, 5×SSCP (SSCcontaining 25 mM phosphate buffer, pH 6.5), 200 μg/ml denatured salmonsperm DNA, 3×Denhardt's, and 10% dextran sulfate. Followinghybridization, the filters were washed extensively at room temperaturein 2×SSC, then for 15 minutes at 47-48° C. Following an exposure toX-ray film, the filters were treated to increasingly stringent washconditions followed by film recording until a final wash with 0.1×SSC at65° C. was reached. Film analysis showed that a “family” of plaques thathybridized at lower wash stringency but not at the highest stringency.This “family” was selected for further analysis.

[0099] Two λ phage clones from the “family” obtained from the initialscreening were subcloned into the Not I site of the pUCtype plasmidvector pBluescript SK⁺ (obtained from Stratagene Cloning Systems, LaJolla,. Calif.) and were analyzed by restriction mapping and sequenceanalysis. Restriction enzyme analysis of a phage clone, designated α1-1,revealed a restriction fragment pattern dissimilar from that of the PDGFβ-receptor cDNA with the exception of a common Bgl II-Bgl II band ofapproximately 160 bp. The PDGF β-receptor cDNA contains two similarlyspaced Bgl II sites within the region coding for the second tyrosinekinase domain.

[0100] Restriction analysis of a second plasmid subclone (designatedα1-7) revealed an overlap of the 5′ approximately 1.2 kb of clone α1-1,and an additional approximately 2.2 kb of sequence extending in the 5′direction. Sequence analysis revealed that the 3′ end of this cloneencodes the second tyrosine kinase domain, which contains regions ofnear sequence identity to the corresponding regions in the PDGFβ-receptor. The 5′ end of clone α1-7 contained non-receptor sequences.Two additional α-receptor clones were obtained by probing with α1-1sequences. Clone α1-1 was digested with Not I and Spe I, and a 230 bpfragment was recovered. Clone α1-1 was also digested with Bam HI and NotI, and a 550 bp fragment was recovered. A clone that hybridized to the230 bp probe was designated α5-1. This clone contained the 5′-mostcoding sequence for the PDGF α-receptor. Another clone, designated α6-3,hybridized to the 550 bp probe and was found to contain 3′ coding andnon-coding sequences, including the poly(A) tail.

[0101] Clone α1-1 was radiolabeled (³²P) and used to probe a northernblot (Thomas, Methods Enzymol. 100: 225-265, 1983) of the MG-63 poly(A)+RNA used to prepare the cDNA library. A single band of approximately 6.6kb was observed. RNA prepared from receptor-positive cell linesincluding the human fibroblast SK4, WI-38 and 7573 cell lines; the mousefibroblast line DI 3T3; the U2-OS human osteosarcoma cell line andbaboon aortic smooth muscle cells, and RNA prepared fromreceptor-negative lines including A431 (an epithelial cell line) and VA13 (SV40-transformed WI-38 cells) were probed by northern format withthe α1-1 cDNA. In all cases, the amount of the 6.6 kb band detected inthese RNA correlated well with the relative levels of α-receptordetected on the respective cell surfaces. The 6.6 kb RNA was notdetected in RNA prepared from any tested cell line of hematopoieticorigin, in agreement with a lack of PDGF α-receptor protein detected onthese cell types.

[0102] Clones α1-1 and and α1-7 were joined at a unique Pst I site inthe region encoding the transmembrane portion of the receptor. Cloneα1-1 was digested with Xba I and Pst I and the receptor sequencefragment was recovered. Clone α1-7 was digested with Pst I and Bam HIand the receptor fragment was recovered. The two fragments were ligatedwith Xba I+Bam HI-digested pIC19R (Marsh et al. Gene 32: 481-486, 1984)to construct plasmid pα17R (FIG. 12).

[0103] The remainder of the 5′-most α-receptor sequence was obtainedfrom clone α5-1 as an Sst I-Cla I fragment. This fragment was joined tothe Eco RI-Sst I receptor fragment of pα17R and cloned into Eco RI+ClaI-digested pBluescript SK+ plasmid to construct plasmid pα17B (FIG. 12).FIG. 11 (Sequence ID Numbers 35 and 36) shows the nucleotide sequenceand deduced amino acid sequence of the cDNA present in pα17B.

Example 2 Construction of a SUC2 Signal Sequence-PDGF β-Receptor Fusion

[0104] To direct the PDGF β-receptor into the yeast secretory pathway,the PDGF β-receptor cDNA was joined to a sequence encoding theSaccharomyces cerevisiae SUC2 signal sequence. Oligonucleotides ZC1453and ZC1454 (Sequence ID Numbers 10 and 11; Table 1) were designed toform an adapter encoding the SUC2 secretory signal flanked by a 5′ EcoRI adhesive end and a 3′ Bgl II adhesive end. ZC1453 and ZC1454 wereannealed under conditions described by Maniatis et al. (ibid.). PlasmidpR-RX1 was digested with Bgl II and Sst II to isolate the 1.7 kbfragment comprising the PDGF β-receptor coding sequence from amino acids28 to 596. Plasmid pR-RX1 was also cut with Sst II and Hind III toisolate the 1.7 kb fragment comprising the coding sequence from aminoacids 597 through the translation termination codon and 124 bp of 3′untranslated DNA. The two 1.7 kb pR-RX1 fragments and the ZC1453/ZC1454adapter were joined with pUC19, which had been linearized by digestionwith Eco RI and Hind III. The resultant plasmid, comprising the SUC2signal sequence fused in-frame with the PDGF β-receptor cDNA, wasdesignated pBTL10 (FIG. 2).

Example 3 Construction of pCBS22

[0105] The BAR1 gene product, Barrier, is an exported protein that hasbeen shown to have three domains. When used in conjunction with a firstsignal sequence, the third domain of Barrier protein has been shown toaid in the secretion of proteins into the medium (MacKay et al., U.S.patent application Ser. No. 104,316).

[0106] The portion-of the BAR1 gene encoding the third domain of Barrierwas joined to a sequence encoding the C-terminal portion of substance P(subP; Munro and Pelham, EMBO J. 3: 3087-3093, 1984). The presence ofthe substance P amino acids on the terminus of the fusion proteinallowed the protein to be detected using commercially availableanti-substance P antibodies. Plasmid pZV9 (deposited as a transformantin E. colistrain RR1, ATCC accession no. 53283), comprising the entireBAR1 coding region and its associated flanking regions, was cut with SalI and Bam HI to isolate the 1.3 kb BAR1 fragment. This fragment wassubcloned into pUC13, which had been cut with Sal I and Bam HI, togenerate the plasmid designated pZV17. Plasmid pZV17 was digested withEco RI to remove the 3′-most 0.5 kb of the BAR1 coding region. Thevector-BAR1 fragment was religated to create the plasmid designatedpJH66 (FIG. 3). Plasmid pJH66 was linearized with Eco RI and blunt-endedwith DNA polymerase I (Klenow fragment). Kinased Bam HI linkers (5′ CCGGAT CCG G 3′) were added and excess linkers were removed by digestionwith Bam HI before religation. The resultant plasmid was designated pSW8(FIG. 3).

[0107] Plasmid pSW81, comprising the TPI1 promoter, the BAR1 codingregion fused to the coding region of the C-terminal portion of substanceP (Munro and Pelham, EMBO J. 3: 3087-3093, 1984) and the TPI1terminator, was derived from pSW8. Plasmid pSW8 was cut with Sal I andBam HI to isolate the 824 bp fragment encoding amino acids 252 through526 of BAR1. Plasmid pPM2, containing the synthetic oligonucleotidesequence encoding the dimer form of the C-terminal portion of substanceP (subP) in M13mp8, was obtained from Hugh Pelham (MRC Laboratory ofMolecular Biology, Cambridge, England). Plasmid pPM2 was linearized bydigestion with Bam HI and Sal I and ligated with the 824 bp BAR1fragment from pSW8. The resultant plasmid, pSW14, was digested with SalI and Sma I to isolate the 871 bp BAR1-substance P fragment. PlasmidpSW16, comprising a fragment of BAR1 encoding amino acids 1 through 250,was cut with Xba I and Sal I to isolate the 767 bp BAR1 fragment. Thisfragment was ligated with the 871 bp BAR1-substance P fragment in athree-part ligation with pUC18 cut with Xba I and Sma I. The resultantplasmid, designated pSW15, was digested with Xba I and Sma I to isolatethe 1.64 kb BAR1-substance P fragment. The ADH1 promoter was obtainedfrom pRL029. Plasmid pRL029, comprising the ADH1 promoter and the BAR15′ region encoding amino acids 1 to 33 in pUC18, was digested with Sph Iand Xba I to isolate the 0.42 kb ADH1 promoter fragment. The TPI1terminator (Alber and Kawasaki, ibid.) was provided as a linearizedfragment containing the TPI1 terminator and pUC18 with a Klenow-filledXba I end and an Sph I end. This fragment was ligated with the 0.42 kbADH1 promoter fragment and the 1.64 kb BAR1-substance P fragment in athree-part ligation to produce plasmid pSW22.

[0108] The ADH1 promoter and the coding region of BAR1, from thetranslation initiation ATG through the Eco RV site present in pSW22,were removed by digestion with Hind III and Eco RV. The 3.9 kb vectorfragment, comprising the 401 bp between the Eco RV and the Eco RI sitesof the BAR1 gene fused to subP and the TPI1 terminator, was isolated bygel electrophoresis. Oligonucleotide ZC1478 (Sequence ID Number 12;Table 1) was kinased and annealed with oligonucleotide ZC1479 (SequenceID Number 13; Table 1) using conditions described by Maniatis et al.(ibid.). The annealed oligonucleotides formed an adapter comprising aHind III adhesive end and a polylinker encoding Bgl II, Sph I, Nru I andEco RV restriction sites. The ZC1479/ZC1478 adapter was ligated with thegel-purified pSW22 fragment. The resultant plasmid was designated pCBS22(FIG. 3).

Example 4 Construction of pBTL13

[0109] In order to enhance the secretion of the PDGF β-receptor and tofacilitate the identification of the secreted protein, a sequenceencoding the third domain of BAR1 fused to the C-terminal amino acids ofsubstance P was fused in frame with the 5′ 1240 bp of the PDGFβ-receptor. Plasmid pBTL10 (Example 2) was digested with Sph I and Sst Ito isolate the 4 kb fragment comprising the SUC2 signal sequence, aportion of the PDGF β-receptor cDNA and the pUC19 vector sequences.Plasmid pCBS22 was digested with Sph I and Sst I to isolate the 1.2 kbfragment comprising the BAR1-subP fusion and the TPI1 terminator. Thesetwo fragments were ligated, and the resultant plasmid was designatedpBTL13 (FIG. 4).

Example 5 Construction of an Expression Vector Encoding the Entire PDGFβ-Receptor

[0110] The entire PDGF β-receptor was directed into the secretorypathway by fusing a SUC2 signal sequence to the 5′ end of the PDGFβ-receptor coding sequence. This fusion was placed behind the TPI1promoter and inserted into the vector YEp13 for expression in yeast.

[0111] The TPI1 promoter was obtained from plasmid pTPIC10 (Alber andKawasaki, J. Mol. Appl. Genet. 1: 410-434, 1982), and plasmid pFATPOT(Kawasaki and Bell, EP 171,142; ATCC 20699). Plasmid pTPIC10 was cut atthe unique Kpn I site, the TPI1 coding region was removed with Bal-31exonuclease, and an Eco RI linker (sequence: GGA ATT CC) was added tothe 3′ end of the promoter. Digestion with Bgl II and Eco RI yielded aTPI1 promoter fragment having Bgl II and Eco RI sticky ends. Thisfragment was then joined to plasmid YRp7′ (Stinchcomb et al., Nature 28239-43, 1979) that had been cut with Bgl II and Eco RI (partial). Theresulting plasmid, TE32, was cleaved with Eco RI (partial) and Bam HI toremove a portion of the tetracycline resistance gene. The linearizedplasmid was then recircularized by the addition of an Eco RI-Bam HIlinker to produce plasmid TEA32. Plasmid TEA32 was digested with Bgl IIand Eco RI, and the 900 bp partial TPI1 promoter fragment wasgel-purified. Plasmid pIC19H (Marsh et al., Gene 32:481-486, 1984) wascut with Bgl II and Eco RI and the vector fragment was gel purified. TheTPI1 promoter fragment was then ligated to the linearized pIC19H and themixture was used to transform E. coli RR1. Plasmid DNA was prepared andscreened for the presence of a ˜900 bp Bgl II-Eco RI fragment. A correctplasmid was selected and designated pICTPIP.

[0112] The TPI1 promoter was then subcloned to place convenientrestriction sites at its ends. Plasmid pIC7 (Marsh et al., ibid.) wasdigested with Eco RI, the fragment ends were blunted with DNA polymeraseI (Klenow fragment), and the linear DNA was recircularized using T4 DNAligase. The resulting plasmid was used to transform E. coli RR1. PlasmidDNA was prepared from the transformants and was screened for the loss ofthe Eco RI site. A plasmid having the correct restriction pattern wasdesignated pIC7RI*. Plasmid pIC7RI* was digested with Hind III and NarI, and the 2500 bp fragment was gel-purified. The partial TPI1 promoterfragment (ca. 900 bp) was removed from pICTPIP using Nar I and Sph I andwas gel-purified. The remainder of the TPI1 promoter was obtained fromplasmid pFATPOT by digesting the plasmid with Sph I and Hind III, and a1750 bp fragment, which included a portion of the TPI1 promoter fragmentfrom pICTPIP, and the fragment from pFATPOT were then combined in atriple ligation to produce pMVR1 (FIG. 2).

[0113] The TPI1 promoter was then joined to the SUC2-PDGF β-receptorfusion. Plasmid pBTL10 (Example 2) was digested with Eco RI and Hind IIIto isolate the 3.4 kb fragment comprising the SUC2 signal sequence andthe entire PDGF β-receptor coding region. Plasmid pMVR1 was digestedwith Bgl II and Eco RI to isolate the 0.9 kb TPI1 promoter fragment. TheTPI1 promoter fragment and the fragment derived from pBTL10 were joinedwith YEp13, which had been linearized by digestion with Bam HI and HindIII, in a three-part ligation. The resultant plasmid was designatedpBTL12 (FIG. 2).

Example 6 Construction of an Expression Vector Encoding the 5′Extracellular Portion of the PDGF β-Receptor

[0114] The extracellular portion of the PDGF β-receptor was directedinto the secretory pathway by fusing the coding sequence to the SUC2signal sequence. This fusion was placed in an expression vector behindthe TPI1 promoter. Plasmid pBTL10 (Example 2) was digested with Eco RIand Sph I to isolate the approximately 1.3 kb fragment comprising theSUC2 signal sequence and the PDGF β-receptor extracellular domain codingsequence. Plasmid pMVR1 (Example 5) was digested with Bgl II and Eco RIto isolate the 0.9 kb TPI1 promoter fragment. The TPI1 promoter fragmentwas joined with the fragment derived from pBTL10 and YEp13, which hadbeen linearized by digestion with Bam HI and Sph I, in a three-partligation. The resultant plasmid was designated pBTL11 (FIG. 2).

Example 7 Construction of Yeast Expression Vectors pBTL14 and pBTL15,and The Expression of PDGF β-Receptor-BAR1-subP Fusions

[0115] A. Construction of pBTL14

[0116] The SUC2-PDGFβ-R fusion was joined with the third domain of BAR1to enhance the secretion of the receptor, and the expression unit wascloned into a derivative of YEp13 termed pJH50. YEp13 was modified todestroy the Sal I site near the LEU2 gene. This was achieved bypartially digesting YEp13 with Sal I followed by a complete digestionwith Xho I. The 2.0 kb Xho I-Sal I fragment comprising the LEU2 gene andthe 8.0 kb linear YEp13 vector fragment were isolated and ligatedtogether. The ligation mixture was transformed into E. coli strain RR1.DNA was prepared from the transformants and was analyzed by digestionwith Sal I and Xho I. A clone was isolated which showed a single Sal Isite and an inactive Xho I site indicating that the LEU2 fragment hadinserted in the opposite orientation relative to the parent plasmidYEp13. The plasmid was designated pJH50.

[0117] Referring to FIG. 4, plasmid pBTL12 (Example 5) was digested withSal I and Pst I to isolate the 2.15 kb fragment comprising 270 bp ofYEp13 vector sequence, the TPI1 promoter, the SUC2 signal sequence, and927 bp of PDGF β-receptor cDNA. Plasmid pBTL13 (Example 4) was digestedwith Pst I and Hind III to isolate the 1.48 kb fragment comprising 313bp of PDGF β-receptor cDNA, the BAR1-subP fusion and the TPI1terminator. The fragments derived from pBTL12 and pBTL13 were joinedwith pJH50, which had been linearized by digestion with Hind III and SalI, in a three-part ligation. The resultant plasmid was designatedpBTL14.

[0118] B. Construction of pBTL15

[0119] Referring to FIG. 5, a yeast expression vector was constructedcomprising the TPI1 promoter, the SUC2 signal sequence, 1.45 kb of PDGFβ-receptor cDNA sequence fused to the BAR1-subP fusion and the TPI1terminator. Plasmid pBTL12 (Example 5) was digested with Sal I and Fsp Ito isolate the 2.7 kb fragment comprising the TPI1 promoter, the SUC2signal sequence, the PDGFβ-R coding sequence, and 270 bp of YEp13 vectorsequence. Plasmid pBTL13 (Example 4) was digested with Nru I and HindIII to isolate the 1.4 kb fragment comprising the BAR1-subP fusion, theTPI1 terminator and 209 bp of 3′ PDGF β-receptor cDNA sequence. Thefragments derived from pBTL12 and pBTL13 were joined in a three-partligation with pJH50, which had been linearized by digestion with HindIII and Sal I. The resultant plasmid was designated pBTL15.

[0120] C. Expression of PDGFβ-R-subP Fusions from pBTL14 and pBTL15

[0121] Yeast expression vectors pBTL14 and pBTL15 were transformed intoSaccharomyces cerevisiae strains ZY100 (MATa leu2-3,112 ade2-101 suc2-Δ9gal2 pep4::TPI1prom-CAT) and ZY400 (MATa leu2-3,112 ade2-101 suc2-Δ9gal2 pep4::TPI1prom-CAT Δmnn9::URA3). Transformations were carried outusing the method essentially described by Beggs (ibid.). Transformantswere selected for their ability to grow on −LEUDS (Table 2). TABLE 2Media Recipes -LeuThrTrp Amino Acid Mixture 4 g adenine 3 g L-arginine 5g L-aspartic acid 2 g L-histidine free base 6 g L-isoleucine 4 gL-lysine-mono hydrochloride 2 g L-methionine 6 g L-phenylalanine 5 gL-serine 5 g L-tyrosine 4 g uracil 6 g L-valine

[0122] Mix all the ingredients and grind with a mortar and pestle untilthe mixture is finely ground. -LEUDS 20 g glucose 6.7 g Yeast NitrogenBase without amino acids (DIFCO Laboratories Detroit, MI) 0.6 g-LeuThrTrp Amino Acid Mixture 182.2 g sorbitol 18 g Agar

[0123] Mix all the ingredients in distilled water. Add distilled waterto a final volume of 1 liter. Autoclave 15 minutes. After autoclavingadd 150 mg L-threonine and 40 mg L-tryptophan. Pour plates and allow tosolidify. -LEUDS + sodium succinate, pH 6.5 20 g Yeast Nitrogen Basewithout amino acids 0.6 g -LeuTrpThr Amino Acid Mixture 182.2 g sorbitol11.8 g succinic acid

[0124] Mix all ingredients in distilled water to a final volume of 1liter. Adjust the pH of the solution to pH 6.5. Autoclave 15 minutes.After autoclaving add 150 mg L-threonine and 40 mg L-tryptophan.Fermentation Medium 7 g/l yeast nitrogen base without amino acids orammonium sulfate (DIFCO Laboratories) 0.6 g/l ammonium sulfate 0.5 Msorbitol 0.39 g/l adenine sulfate 0.01% polypropylene glycol

[0125] Mix all ingredients in distilled water. Autoclave 15 minutes. Add80 ml 50% glucose for each liter of medium. Super Synthetic -LEUD, pH6.5 (liquid or solid medium) 6.7 g Yeast Nitrogen Base without aminoacids or ammonium sulfate (DIFCO) 6 g ammonium sulfate 160 g adenine 0.6g -LeuThrTrp Amino Acid Mixture 20 g glucose 11.8 g succinic acid

[0126] Mix all ingredients and add distilled water to a final volume of800 ml. Adjust the pH of the solution to pH 6.4. Autoclave 15 minutes.For solid medium, add 18 g agar before autoclaving, autoclave and pourplates.

[0127] Super Synthetic-LEUDS, pH 6.4 (Liquid or Solid Medium)

[0128] Use the same recipe as Super Synthetic −LEUD, pH 6.4, but add182.2 g sorbitol before autoclaving. YEPD 20 g glucose 20 g BactoPeptone (DIFCO Laboratories) 10 g Bacto Yeast Extract (DIFCOLabloratories) 18 g agar  4 ml adenine 1%  8 ml 1% L-leucine

[0129] Mix all ingredients in distilled water, and bring to a finalvolume of 1 liter. Autoclave 25 minutes and pour plates.

[0130] The transformants were assayed for binding to an anti-PDGFβ-receptor monoclonal antibody (PR7212) or an anti-substance P antibodyby protein blot assay. ZY100[pBTL14] and ZY100[pBTL15] transformantswere grown overnight at 30° C. in 5 ml Super Synthetic −LEUD, pH 6.4(Table 2). ZY400[pBTL14] and ZY400[pBTL15] transformants. were grownovernight at 30° C. in 5 ml Super Synthetic-LEUDS, pH 6.4 (Table 2). Thecultures were pelleted by centrifugation and the supernatants wereassayed for the presence of secreted PDGF β-receptor analogs by proteinblot assay using methods described in Example 18. Results of assaysusing PR7212 are shown in Table 3. TABLE 3 Results of a protein blotprobed with PR7212 Transformant: ZY100[pBTL14] + ZY400[pBTL14] ++ZY100[pBTL15] + ZY400[pBTL15] +

Example 8 Construction of a SUC2-PDGFβ-R Fusion Comprising the CompletePDGFβ-R Extracellular Domain

[0131] A. Construction of pBTL22

[0132] The PDGFβ-R coding sequence present in pBTL10was modified todelete the coding region 3′ to the extracellular PDGFβ-R domain. Asshown in FIG. 6, plasmid pBTL10 was digested with Sph I and Bam HI andwith Sph I and Sst II to isolate the 4.77 kb fragment and the 466 bpfragment, respectively. The 466 bp fragment was then digested with Sau3A to isolate the 0.17 kb fragment. The 0.17 kb fragment and the 4.77 kbwere joined by ligation. The resultant plasmid was designated pBTL21.

[0133] Plasmid pBTL21, containing a Bam HI site that was regenerated bythe ligation of the Bam HI and Sau 3A sites, was digested with Hind IIIand Bam HI to isolate the 4.2 kb fragment. Synthetic oligonucleotidesZC1846 (Sequence ID Number 16; Table 1) and ZC1847 (Sequence ID Number17; Table 1) were designed to form an adapter encoding the PDGFβ-R fromthe Sau 3A site after bp 1856 (FIG. 1; (Sequence ID Number 1)) to theend of the extracellular domain at 1958 bp (FIG. 1; Sequence ID Number1), having a 5′ Bam HI adhesive end that destroys the Bam HI site and a3′ Hind III adhesive end. oligonucleotides ZC1846 and ZC1847 wereannealed under conditions described by Maniatis et. al. (ibid.). The 4.2pBTL21 fragment and the ZC1846/ZC1847 adapter were joined by ligation.The resultant plasmid, designated pBTL22, comprises the SUC2 signalsequence fused in proper reading frame to the extracellular domain ofPDGFβ-R in the vector pUC19 (FIG. 6).

[0134] B. Construction of pBTL28

[0135] An in-frame translation stop codon was inserted immediately afterthe coding region of the PDGFβ-R in pBTL22 using oligonucleotides ZC1892(Sequence ID Number 19; Table 1) and ZC1893 (Sequence ID Number 20;Table 1). These oligonucleotides were designed to form an adapterencoding a stop codon in-frame with the PDGFβ-R coding sequence frompBTL22 flanked by a 5′ Hind III adhesive end and a 3′ Xba I adhesiveend. Plasmid pBTL22 was digested with Eco RI and Hind III to isolate the1.6 kb SUC2-PDGFβ-R fragment. Plasmid pMVR1 was digested with Eco RI andXba I to isolate the 3.68 kb fragment comprising the TPI1 promoter,pIC7RI* vector sequences and the TPI1 terminator. OligonucleotidesZC1892 and ZC1893 were annealed to form a Hind III-Xba I adapter. The1.6 kb SUC2-PDGFβ-R fragment, the 3.86 kb pMVR1 fragment and theZC1892/ZC1893 adapter were joined in a three-part ligation. Theresultant plasmid was designated pBTL27.

[0136] The expression unit present in pBTL27 was inserted into the yeastexpression vector pJH50 by first digesting pJH50 with Bam HI and Sal Ito isolate the 10.3 kb vector fragment. Plasmid pBTL27 was digested withBgl II and Eco RI and with Xho I and Eco RI to isolate the 0.9 kb TPI1promoter fragment and the 1.65 kb fragment, respectively. The 10.3 kbpJH50 vector fragment, the 0.9 kb TPI1 promoter fragment and 1.65 kbfragment were joined in a three-part ligation. The resultant plasmid wasdesignated pBTL28.

[0137] C. Construction of Plasmid pBTL30

[0138] The PDGFβ-R coding sequence present in plasmid pBTL22 wasmodified to encode the twelve C-terminal amino acids of substance P andan in-frame stop codon. Plasmid pBTL22 was digested with Eco RI and HindIII to isolate the 1.6 kb SUC2-PDGFβ-R fragment. Plasmid pMVR1 wasdigested with Eco RI and Xba I to isolate the 3.68 kb fragmentcomprising the TPI1 promoter, pIC7RI* and the TPI1 terminator. Syntheticoligonucleotides ZC1894 (Sequence ID Number 21; Table 1) and ZC1895(Sequence ID Number 22; Table 1) were annealed to form an adaptercontaining the codons for the twelve C-terminal amino acids of substanceP followed by an in-frame stop codon and flanked on the 5′ end with aHind III adhesive end and on the 3′ end with an Xba I adhesive end. TheZC1894/ZC1895 adapter, the 1.6 kb SUC2-PDGFβ-R fragment and the pMVR1fragment were joined in a three-part ligation. The resultant plasmid,designated pBTL29, was digested with Eco RI and Xho I to isolate the1.69 kb SUC2-PDGFβ-R-subP-TPI1 terminator fragment. Plasmid pBTL27 wasdigested with Bgl II and Eco RI to isolate the 0.9 kb TPI1 promoterfragment. Plasmid pJH50 was digested with Bam HI and Sal I to isolatethe 10.3 kb vector fragment. The 1.69 kb pBTL29 fragment, the 0.9 kbTPI1 promoter fragment and the 10.3 kb vector fragment were joined in athree-part ligation. The resulting plasmid was designated pBTL30.

Example 9 Construction and Expression of a SUC2-PDGFβ-R-IgG HingeExpression Vector

[0139] An expression unit comprising the TPI1 promoter, the SUC2 signalsequence, the PDGFβ-R extracellular domain, an immunoglobulin hingeregion and the TPI1 terminator was constructed. Plasmid pBTL22 wasdigested with Eco RI and Hind III to isolate the 1.56 kb fragmentsPlasmid pMVR1 was digested with Eco RI and Xba I to isolate the 3.7 kbfragment, comprising the TPI1 promoter, pIC7RI* vector sequences and theTPI1 terminator. Oligonucleotides ZC1776 (Sequence ID Number 14;Table 1) and ZC1777 (Sequence ID Number 15; Table 1) were designed toform, when annealed, an adapter encoding an immunoglobulin hinge regionwith a 5′ Hind III adhesive end and a 3′ Xba I adhesive end.Oligonucleotides ZC1776 and ZC1777 were annealed under conditionsdescribed by Maniatis et al. (ibid.). The 1.56 kb pBTL22 fragment, the3.7 kb fragment and the ZC1776/ZC1777 adapter were joined in athree-part ligation, resulting in plasmid pBTL24.

[0140] The expression unit of pBTL24, comprising the TPI1 promoter, SUC2signal sequence, PDGFβ-R extracellular domain sequence, hinge regionsequence, and TPI1 terminator, was inserted into pJH50. Plasmid pBTL24was digested with Xho I and Hind III to isolate the 2.4 kb expressionunit. Plasmid pJH50 was digested with Hind III and Sal I to isolate the9.95 kb fragment. The 2.4 kb pBTL24 fragment and 9.95 kb pJH50 vectorfragment were joined by ligation. The resultant plasmid was designatedpBTL25.

[0141] Plasmid pBTL25 was transformed into Saccharomyces cerevisiaestrain ZY400 using the method essentially described by Beggs (ibid.).Transformants were selected for their ability to grow on −LEUDS (Table2). The transformants were tested for their ability to bind theanti-PDGFβ-R monoclonal antibody PR7212 using the colony assay methoddescribed in Example 18. Plasmid pBTL25 transformants were patched ontonitrocellulose filters that had been wetted and supported by YEPD solidmedium. Antibody PR7212 was found to bind to the PDGFβ-R-IgG hingefusion secreted by ZY400[pBTL25] transformants.

Example 10 Construction and Expression of a SUC2 Signal Sequence-PDGFβ-RExtracellular Domain-SUC2 Fusion

[0142] As shown in FIG. 6, an expression unit comprising the TPI1promoter, SUC2 signal sequence, PDGFβ-R extracellular domain sequence,and SUC2 coding sequence was constructed as follows. Plasmid pBTL22 wasdigested with Eco RI and Hind III to isolate the 1.6 kb SUC2-PDGFβ-Rfragment. Plasmid pMVR1 was digested with Bgl II and Eco RI to isolatethe 0.9 kb TPI1 promoter fragment. The SUC2 coding region was obtainedfrom pJH40. Plasmid pJH40 was constructed by inserting the 2.0 kb HindIII-Hind III SUC2 fragment from pRB58 (Carlson et al., Cell 28: 145-154,1982) into the Hind III site of pUC19 followed by the destruction of theHind III site 3′ to the coding region. Plasmid pJH40 was digested withHind III and Sal I to isolate the 2.0 kb SUC2 coding sequence. PlasmidpJH50 was digested with Sal I and Bam HI to isolate the 10.3 kb vectorfragment. The 0.9 kb Bgl II-Eco RI TPI1 promoter fragment, the 1.6 kbEco RI-Hind III SUC2-PDGFβ-R, the 2.0 kb Hind III-Sal I SUC2 fragmentand the 10.3 kb Bam HI-Sal I vector fragment were joined in a four-partligation. The resultant plasmid was designated pBTL26 (FIG. 6).

[0143] Plasmid pBTL26 was transformed into Saccharomyces cerevisaestrain ZY400 using the method essentially described by Beggs (ibid.).Transformants were selected for their ability to grow on −LEUDS (Table2). ZY400 transformants (ZY400[pBTL26]) were assayed by protein blot(Example 18), colony blot (Example 18) and competition assay.

[0144] Protein blot assays were carried out on ZY400[pBTL26] andZY400[pJH50]) (control) transformants that had been grown in flasks. Twohundred-fifty microliters of a 5 ml overnight cultures of ZY400[pBTL26]and ZY400 [pJH50] in −LEUDS+sodium succinate, pH 6.5 (Table 2) wereinoculated into 50 ml of −LEUDS+sodium succinate, pH 6.5. The cultureswere incubated for 35 hours in an airbath shaker at 30° C. The culturesupernatants were harvested by centrifugation. The culture supernatantswere assayed as described in Example 18 and were found to bind PR7212antibody.

[0145] Colony assays were carried out on ZY400[pBTL26] transformants.ZY400[pBTL26] transformants were patched onto wetted nitrocellulosefilters that were supported on a YEPD plate. The colony assay carriedout as described in Example 8.A showed that ZY400[pBTL26] antibodiesbound PR7212 antibodies.

[0146] Competition binding assays were carried out on ZY400[pBTL26] andZY400[pJH50] transformants. The transformants were grown in two litersof fermentation medium (Table 2) in a New Brunswick Bioflo2 fermentor(New Brunswick, Philadelphia, Pa.) with continuous pH control at pH 6.4.The cultures were adjusted to pH 7.5 immediately prior to harvesting.Culture supernatants were concentrated in an Amicon concentrator(Amicon, San Francisco, Calif.) using an Amicon 10⁴ mw spiral filtercartridge. The concentrated supernatants were further concentrated usingAmicon Centriprep 10's. Fifteen milliliters of the concentratedsupernatant samples were added to the Centripreps, and the. Centriprepswere spun in a Beckman GRP centrifuge (Beckman Instruments Inc.,Carlsbad, Calif.) at setting 5 for a total of 60 minutes. Theconcentrates were removed from the Centripreps and were assayed in thecompetition assay.

[0147] The competition binding assay measured the amount of ¹²⁵I-PDGFleft to bind to fetal foreskin fibroblast cells after preincubation withthe concentrate containing the PDGFβ-R-SUC2 fusion protein. PDGF-AA andPDGF-AB were iodinated using the Iodopead method (Pierce Chemical).PDGF-BB_(Tyr) was iodinated and purified as described in Example 18.F.The concentrate was serially diluted in binding medium (Table 4). Thedilutions were mixed with 0.5 ng of iodinated PDGF-AA, PDGF-BBTyr orPDGF-AB, and the mixtures were incubated for two hours at roomtemperature. Three hundred micrograms of unlabeled PDGF-BB was added toeach sample mixture. The sample mixtures were added to 24-well platescontaining confluent fetal foreskin fibroblast cells (AG1523, availablefrom the Human Genetic Mutant Cell Repository, Camden, N.J.). The cellswere incubated with the mixture for four hours at 4° C. The supernatantswere aspirated from the wells, and the wells were rinsed three timeswith phosphate buffered saline that was held at 4° C. (PBS; Sigma, St.Louis, Mo.). Five hundred microliters of PBS+1% NP-40 was added to eachwell, and the plates were shaken on a platform shaker for five minutes.The cells were harvested and the amount of iodinated PDGF wasdetermined. The results of the competition binding assay showed that thePDGFβ-R-SUC2 fusion protein was able to competitively bind all threeisoforms of PDGF.

[0148] The PDGFβ-R produced from ZY400 [pBTL26] transformants was testedfor cross reactivity to fibroblast growth factor (FGF) and transforminggrowth factor-β (TGF-β) using the competition assay essentiallydescribed above. Supernatant concentrates from ZY400[pBTL26] andZY400[JH50] (control) transformants were serially diluted in bindingmedium (Table 4). The dilutions were mixed with 7.9 ng of iodinated FGFor 14 ng of iodinated TGF-β, and the mixtures were incubated for twohours at room temperature. Fourteen micrograms of unlabeled FGF wasadded to each mixture containing labeled FGF, and 7 μg of unlabeledTGF-β was added to each mixture containing labeled TGF-β. The samplemixtures were added to 24-well plates containing confluent human dermalfibroblast cells. (Human dermal fibroblast cells express both FGFreceptors and TGFβ receptors.) The cells were incubated with themixtures for four hours at 4° C. Five hundred microliters of PBS+1%NP-40 was added to each well, and the plates were shaken on a platformshaker for five minutes. The cells were harvested and the amount ofiodinated FGF or TGF-β bound to the cells was determined.

[0149] The results of these assays showed that the PDGFβ-R-SUC2 fusionprotein did not cross react with FGF or TGF-β. TABLE 4 Reagent RecipesBinding Medium 500 ml Ham's F-12 medium 12 ml 1 M HEPES, pH 7.4 5 ml100x PSN (Penicillin/Streptomycin/ Neomycin, Gibco) 1 g rabbit serumalbumin Western Transfer Buffer 25 mM Tris, pH 8.3 19 mM glycine, pH 8.320% methanol Western Buffer A 50 ml 1 M Tris, pH 7.4 20 ml 0.25 mM EDTA,pH 7.0 5 ml 10% NP-40 37.5 ml 4 M NaCl 2.5 g gelatin

[0150] The Tris, EDTA, NP-40 and NaCl were diluted to a final volume ofone liter with distilled water. The gelatin was added to 300 ml of thissolution and the solution was heated in a microwave until the gelatinwas in solution. The gelatin solution was added back to the remainder ofthe first solution and stirred at 4 ° C. until cool. The buffer wasstored at 4 ° C. Western Buffer B 50 ml 1 M Tris, pH 7.4 20 ml 0.25 MEDTA, pH 7.0 5 ml 10% NP-40 58.4 g NaCl 2.5 g gelatin 4 g N-lauroylsarcosine

[0151] The Tris, EDTA, NP-40, and the NaCl were mixed and diluted to afinal volume of one liter. The gelatin was added to 300 ml of thissolution and heated in a microwave until the gelatin was in solution.The gelatin solution was added back to the original solution and theN-lauroyl sarcosine was added. The final mixture was stirred at 4° C.until the solids were completely dissolved. This buffer was stored at 4° C. 2x Loading Buffer 36 ml 0.5 M Tris-HCl, pH 6.8 16 ml glycerol 16 ml20% SDS  4 ml 0.5% Bromphenol Blue in 0.5 M Tris-HCl, pH 6.8

[0152] Mix all ingredients. Immediately before use, add 100 μlβ-mercaptoethanol to each 900 μl dye mix

Example 11 Construction and Expression of PDGF Receptor Analogs From BHKCells

[0153] A. Construction of pBTL114 and pBTL115

[0154] The portions of the PDGF β-receptor extracellular domain presentin pBTL14 and pBTL15 were placed in a mammalian expression vector.Plasmids pBTL14 and pBTL15 were digested with Eco RI to isolate the 1695bp and 1905 bp SUC2 signal-PDGFβ-R-BAR1 fragments. The 1695 bp fragmentand the 1905 bp fragment were each ligated to Zem229R that had beenlinearized by digestion with Eco RI.

[0155] The vector Zem229R was constructed as shown in FIG. 10 fromZem229. Plasmid Zem229 is a pUC18-based expression vector containing aunique Bam HI site for insertion of cloned DNA between the mousemetallothionein-1 promoter and SV40 transcription terminator and anexpression unit containing the SV40 early promoter, mouse dihydrofolatereductase gene, and SV40 transcription terminator. Zem229 was modifiedto delete the Eco RI sites flanking the Bam HI cloning site and toreplace the Bam HI site with a single Eco RI cloning site. The plasmidwas partially digested with Eco RI, treated with DNA polymerase I(Klenow fragment) and dNTPs, and religated. Digestion of the plasmidwith Bam HI followed by ligaion of the linearized plasmid with a BamHI-Eco I adapter resulted in a unique Eco RI cloning site. The resultantplasmid was designated Zem229R.

[0156] The ligation mixtures were transformed into E. coli strain RR1.Plasmid DNA was prepared and the plasmids were subjected to restrictionenzyme analysis. A plasmid having the 1695 bp pBTL14 fragment insertedinto Zem229R in the correct orientation was designated pBTL114 (FIG. 9).A plasmid having the 1905 bp pBTL15 fragment inserted into Zem229R inthe correct orientation was designated pBTL115 (FIG. 9).

[0157] B. Expression of Secreted PDGF β-receptor Analogs in tk⁻ ts13 BHKCells

[0158] Plasmids pBTL114 and pBTL115 were each transfected into tk⁻ts13cells using calcium phosphate precipitation (essentially as described byGraham and van der Eb, J. Gen. Virol. 36: 59-72, 1977). The transfectedcells were grown in Dulbecco's modified Eagle's medium (DMEM) containing10% fetal calf serum, 1×PSN antibiotic mix (Gibco 600-5640), 2.0 mML-glutamine. The cells were selected in 250 nM methotrexate (MTX) for 14days, and the resulting colonies were screened by the immunofilter assay(McCracken and Brown, Biotechniques, 82-87, March/April 1984). Plateswere rinsed with PBS or No Serum medium (DMEM plus 1×PSN antibioticmix). Teflon® mesh (Spectrum Medical Industries, Los Angeles, Calif. )was then placed over the cells. Nitrocellulose filters were wetted withPBS or No Serum medium, as appropriate, and placed over the mesh. Aftersix hours incubation at 37° C., filters were removed and placed inWester buffer A (Table 4) overnight at room temperature. The filterswere developed using the antibody PR7212 and the procedure described inExample 8. The filters showed that conditioned media frompBTL114-transfected and pBTL115-transfected BHK cells bound the PR7212antibody indicating the presence of biologically active secretedPDGFβ-R.

Example 12 Expression of PDGF β-Receptor Analogs in Cultured MouseMyeloma Cells

[0159] A. Construction of pICμPRE8

[0160] The immunoglobulin μ heavy chain promoter and enhancer weresublconed into pIC19H to provide a unique Hind III site 3′ to thepromoter. Plasmid pμ (Grosschedl and Baltimore, Cell 41: 885-897, 1985)was digested with Sal I and Eco RI to isolate the 3.1 kb fragmentcomprising the μ promoter. Plasmid pIC19H was linearized by digestionwith Eco RI and Xho I. The μ promoter fragment and the linearized pIC19Hvector fragment were joined by ligation. The resultant plasmid,designated pICμ3, was digested with Ava II to isolate the 700 bp μpromoter fragment. The 700 bp fragment was blunt-ended by treatment withDNA polymerase I (Klenow fragment) and deoxynucleotide triphosphates.Plasmid pIC19H was linearized by digestion with Xho I, and the adhesiveends were filled in by treatment with DNA polymerase I (Klenowfragement) and deoxynucleotide triphosphates. The blunt-ended Ava IIfragment was ligated with the blunt-ended, linearized pIC19H, and theligation mixture was transformed into E coli JM83. Plasmid DNA wasprepared from the transformants and was analyzed by restriction digest.A plasmid with a Bgl II site 5′ to the promoter was designatedPICμPR1(−). Plasmid pICμPR1(−) was digested with Hind III and Bgl II toisolate the 700 bp μ promoter fragment. Plasmid pIC19R was linearized bydigestion with Hind III and Bam HI. The 700 bp promoter fragment wasjoined with the linearized pIC19R by ligation. The resultant plasmid,designated pICμPR7, comprised the μ promoter with an unique Sma I site5′ to the promoter and a unique Hind III site 3′ to the promoter.

[0161] The immunoglobulin heavy chain μ enhancer (Gillies et al., Cell33: 717-728, 1983) was inserted into the unique Sma I site to generateplasmid pICμPRE8. Plasmid pJ4 (obtained from F. Blattner, Univ.Wisconsin, Madison, Wis.), comprising the 1.5 kb Hind III-Eco RI μenhancer fragment in the vector pAT153 (Amersham, Arlington Heights,Ill.), was digested with Hind III and Eco RI to isolate the 1.5 kbenhancer fragment. The adhesive ends of the enhancer fragment werefilled in by treatment with T4 DNA polymerase and deoxynucleotidetriphosphates. The blunt-ended fragment and pICμPR7, which had beenlinearized by digestion with Sma I, were joined by ligation. Theligation mixture was transformed into E. coli RR1. Plasmid DNA wasprepared from the transformants, and the plasmids were analyzed byrestriction digests. A plasmid comprising the μ enhancer and the μpromoter was designated pICμPRE8 (FIG. 7).

[0162] B. Construction of pSDL114

[0163] The DNA sequence encoding the extracellular domain of the PDGFβ-receptor was joined with the DNA sequence encoding the humanimmunoglobulin light chain constant region. The PDGF β-receptorextracellular domain was obtained from mpBTL22, which comprised the EcoRI-Hind III fragment from pBTL22 (Example 8.A.) cloned into Eco RI-HindIII cut M13mp18. Single stranded DNA was prepared from a mpBTL22 phageclone, and the DNA was subjected to in vitro mutagenesis using theoligonucleotide ZC1886 (Table 1) and the method described by Kunkel(Proc. Natl. Acad. Sci. USA 82: 488-492, 1985). A phage clone comprisingthe mutagenized PDGFβ-R with a donor splice site (5′ splice site) at the3′ end of the PDGFβ-R extracellular domain was designated pBTLR-HX (FIG.7).

[0164] The native PDGFβ-R signal sequence was obtained from pPR5.Plasmid pPR5, comprising 738 bp of 5′ coding sequence with an Eco RIsite immediately 5′ to the translation initiation codon, was constructedby in vitro mutagenesis of the PDGFβ-R cDNA fragment from RP51 (Example1). Replicative form DNA of RP51 was digested with Eco RI to isolate the1.09 kb PDGFβ-R fragment. The PDGFβ-R fragment was cloned into the EcoRI site of M13mp18. Single stranded template DNA was prepared from aphage clone containing the PDGFβ-R fragment in the proper orientation.The template DNA was subjected to in vitro mutagenesis usingoligonucleotide ZC1380 (Sequence ID Number 8; Table 1) and the methoddescribed by Zoller and Smith (Meth. Enzymol. 100: 468-500, 1983). Themutagenesis resulted in the placement of an Eco RI site immediately 5′to the translation initiation codon. Mutagenized phage clones wereanalyzed by dideoxy sequence analysis. A phage clone containing theZC1380 mutation was selected, and replicative form (Rf) DNA was preparedfrom the phage clone. The Rf DNA was digested with Eco RI and Acc I toisolate the 0.63 kb fragment. Plasmid pR-RXI (Example 1) was digestedwith Acc I and Eco RI to isolate the 3.7 kb fragment. The 0.63 kbfragment and the 3.7 kb fragment were joined by ligation resulting inplasmid pPR5 (FIG. 7).

[0165] As shown in FIG. 7, the PDGFβ-R signal peptide and part of theextracellular domain were obtained from plasmid pPR5 as a 1.4 kb EcoRI-Sph I fragment. Replicative form DNA from phage clone pBTLR-HX wasdigested with Sph I and Hind III to isolate the approximately 0.25 kbPDGFβ-R fragment. Plasmid pUC19 was linearized by digestion with Eco RIand Hind III. The 1.4 kb Eco RI-Sph I PDGFβ-R fragment, the 0.25 kb SphI-Hind III fragment from pBTLR-HX and the Eco RI-Hind III cut pUC19 werejoined in a three-part ligation. The resultant plasmid, pSDL110, wasdigested with Eco RI and Hind III to isolate the 1.65 kb PDGFβ-Rfragment.

[0166] Plasmid pICHuCκ3.9.11 was used as the source of the humanimmunoglobulin light chain gene (FIG. 7). The human immunoglobulin lightchain gene was isolated from a human genomic library using anoligonucleotide probe (5′ TGT GAC ACT CTC CTG GGA GTT A 3′; Sequence IDNumber 32), which was based on a published human kappa C gene sequence(Hieter et al., Cell 22: 197-207, 1980). The human light chain (kappa)constant region was subcloned as a 1.1 kb Sph I-Hinf I genomic fragmentof the human kappa gene, which has been treated with DNA polymerase DNAI (Klenow Fragment) to fill in the Hinf I adhesive end, into Sph I-HincII cut pUC19. The 1.1 kb human kappa constant region was susbsequentlyisolated as a 1.1 kb Sph I-Bam HI fragment that was subcloned into SphI-Bgl II cut pIC19R (Marsh et al., ibid.). The resultant plasmid wasdesignated pICHuCλ3.9.11. Plasmid pICHuC_(κ)3.9.11 was digested withHind III and Eco RI to isolate the 1.1 kb kappa constant region gene.Plasmid pIC19H was linearized by digestion with Eco RI. The 1.65 kbPDGFβ-R fragment, the 1.1 kb human kappa constant region fragment andthe linearized pIC19H were joined in a three part ligation. Theresultant plasmid, pSDL112, was digested with Bam HI and Cla I toisolate the 2.75 kb fragment. Plasmid pμPRE8 was linearized with Bgl IIand Cla I. The 2.75 kb fragment and the linearized pμPRE8 were joined byligation. The resultant plasmid was designated pSDL114 (FIG. 7).

[0167] Plasmid pSDL114 was linearized by digestion with Cla I and wascotransfected with Pvu I-digested p416 into SP2/0-Ag14 (ATCC CRL 1581)by electroporation using the method essentially described by Neumann etal. (EMBO J. 1: 841-845, 1982). (Plasmid p416 comprises the Adenovirus 5ori, SV40 enhancer, Adenovirus 2 major late promoter, Adenovirus 2tripartite leader, 5′ and 3′ splice sites, the DHFR^(r) cDNA, the SV40polyadenylation signal and pML-1 (Lusky and Botchan, Nature 293:79-81,1981) vector sequences.) Transfectants were selected in growth mediumcontaining methotrexate.

[0168] Media from drug resistant clones were tested for the presence ofsecreted PDGF β-receptor analogs by enzyme-linked immunosorbant assay(ELISA). Ninety-six well assay plates were prepared by incubating 100 μlof 1 μg/ml polyclonal goat anti-human kappa chain (Cappel Laboratories,Melvern, Pa.) diluted in phosphate buffered saline (PBS; Sigma)overnight at 40° C. Excess antibody was removed by three washes with0.5% Tween 20 in PBS. One hundred microliters of spent media was addedto each well, and the well were incubated for one hour at 4° C. Unboundproteins were removed by eight washes with 0.5% Tween 20 in PBS. Onehundred microliters of peroxidase-conjugated goat anti-human kappaantibody (diluted 1:1000 in a solution containing 5% chicken serum(GIBCO-BRL)+0.5% Tween 20 in PBS) was added to each well and the wellswere incubated for one hour at 4° C. One hundred microliters ofchromophore (100 μl ABTS (2,2′-Azinobis(3-ethylbenz-thiazoline sulfonicacid) diammonium salt; Sigma)+1 μl 30% H₂O₂+12.5 ml citrate/phosphatebuffer (9.04 g/l citric acid, 10.16 g/l Na₂HPO₄)) was added to eachwell, and the wells were incubated to thirty minutes at roomtemperature. The samples were measured at 405 nm. The results of theassay showed that the PDGFβ-R analog secreted by the transfectantscontained an immunoglobulin light chain.

[0169] Spent media from drug resistant clones was also tested for thepresence of secreted PDGF β-receptor analogs by immunoprecipitation.Approximately one million drug resistant transfectants weremetabolically labeled by growth in DMEM medium lacking cysteine+2% calfserum for 18 hours at 37° C. in the presence of 50 μCI ³⁵S-cysteine.Media was harvested from the labeled cells and 250 μl of the spent mediawas assayed by immunoprecipitation with the anti-PDGF β-receptorantibody PR7212 to detect the presence of metabolically labeled PDGFβ-receptor analogs. PR7212, diluted in PBS, was added to the media to afinal concentration of 2.5 μg per 250 μl spent media. Five microlitersof rabbit anti-mouse Ig diluted in PBS was added to the PR7212/mediamixtures. The immunocomplexes were precipitated by the addition of 50 μl10% fixed Staph A (weight/volume in PBS). The immunocomplexes wereanalyzed on 8% SDS-polyacrylamide gels followed by autoradiographyovernight at −70° C. The results of the immunoprecipitation showed thatthe PDGF β-receptor analog secreted by the transfectants was bound bythe anti-PDGF β-receptor antibody. The combined results of the ELISA andimmunoprecipitation assays showed that the PDGF β-receptor analogsecreted by the transfectants contained both the PDGF β-receptorligand-binding domain and the human light chain constant region.

[0170] C. Cotransfection of pSDL114 with an Immunoglobulin Heavy Chain

[0171] Plasmid pSDL114 was cotransfected with pφ5V_(H)huC_(γ)1M-neo,which encodes a neomycin resistance gene expression unit and a completemouse/human chimeric immunoglobulin heavy chain gene expression unit.

[0172] Plasmid pφ5V_(H)huC_(γ)1M-neo was constructed as follows. Themouse immunoglobulin heavy chain gene was isolated from a lambda genomicDNA library constructed from the murine hybridoma cell line NR-ML-05(Serafini et al., Eur. J. Nucl. Med. 14: 232, 1988) using anoligonucleotide probe designed to span the V_(H)/D/J_(H) junction (5′GCA TAG TAG TTA CCA TAT CCT CTT GCA CAG 3′; Sequence ID Number 33). Thehuman immunoglobulin gamma-1 C gene was isolated from a human genomiclibrary using a cloned human gamma-4 constant region gene (Ellison etal., DNA 1: 11-18, 1981). The mouse immunoglobulin variable region wasisolated as a 5.3 kb Sst I-Hind III fragment from the original phageclone and the human gamma-1 C gene was obtained from the original phageclone as a 6.0 kb Hind III-Xho I fragment. The chimeric gamma-1 C genewas created by joining the V_(H) and C_(H) fragments via the common HindIII site and incorporating them with the E. coli neomycin resistancegene expression unit into pIC19H to yield pφ5V_(H)huC_(γ)1M-neo.

[0173] Plasmid pSDL114 was linearized by digestion with Cla I and wasco-transfected into SP2/O-Ag14 cells with Asp 718 linearizedpφ5V_(H)huC_(γ)1M-neo. The transfectants were selected in growth mediumcontaining methotrexate and neomycin. Media from drug-resistant cloneswere tested for their ability to bind PDGF in a competition bindingassay.

[0174] The competition binding assay measured the amount of ¹²⁵I-PDGFleft to bind to human dermal fibroblast cells after preincubation withthe spent media from pSDL114-pφ5V_(H)huC_(γ)1M-neo transfected cells.The media were serially diluted in binding medium (Table 4). Thedilutions were mixed with 0.5 ng of iodinated PDGF-BB or iodinatedPDGF-AA, and the mixtures were incubated for two hours at roomtemperature. Three hundred micrograms of unlabeled PDGF-BB or unlabeledPDGF-AA was added to one tube from each series. The sample mixtures wereadded to 24 well plates containing confluent human dermal fibroblastcells. The cells were incubated with the mixture for four hours at 4° C.The supernatants were aspirated from the wells, and the wells wererinsed three times with phosphate buffered saline that was held a 4° C.(PBS; Sigma, St. Louis, Mo.). Five hundred microliters of PBS+1% NP-40was added to each well, and the plates were shaken on a platform shakerfor five minutes. The cells were harvested and the amount of iodinatedPDGF was determined. The results of the competition binding assay showedthat the protein produced from pSDL114-pφ5V_(H)huC_(γ)1M-neo transfectedcells was able to competitively bind PDGF-BB but did not bind PDGF-AA.

[0175] The PDGF β-receptor analog produced from apSDL114-pφ5V_(H)huC_(γ)1M-neo transfectant was assayed to determine ifthe receptor analog was able to bind PDGF-BB with high affinity. Eightand one half milliliters of spent media containing the PDGFβ-R analogsfrom a pSDL114-pφ5V_(H)huC_(γ)1M-neo transfectant was added to 425 μl ofSepharose Cl-4B-Protein A beads (Sigma, St. Louis, Mo.), and the mixturewas incubated for 10 minutes at 4° C. The beads were pelleted bycentrifugation and washed with binding medium (Table 4). Following thewash the beads were resuspended in 8.5 ml of binding media, and 0.25 mlaliquots were dispensed to 1.5 ml tubes. Binding reactions were preparedby adding iodinated PDGF-BB_(Tyr) (Example 18.F.) diluted in DMEM+10%fetal calf serum to the identical aliquots of receptor-bound beads tofinal PDGF-BB_(Tyr) concentrations of between 4.12 pM and 264 pM.Nonspecific binding was determined by adding a 100 fold excess ofunlabeled BB to an identical set of binding reactions. Mixtures wereincubated overnight at 4° C.

[0176] The beads were pelleted by centrifugation, and unbound PDGF-BBwas removed with three washes in PBS. The beads were resuspended in 100μl of PBS and were counted. Results of the assay showed that the PDGFβ-Ranalog was able to bind PDGF-BB with high affinity.

[0177] D. Construction of pSDL113

[0178] As shown in FIG. 8, the DNA sequence encoding the extracellulardomain of the PDGF β-receptor was joined with the DNA sequence encodinga human immunoglobulin heavy chain constant region joined to a hingesequence. Plasmid pSDL110 was digested with Eco RI and Hind III toisolate the 1.65 kb PDGFβ-R fragment. Plasmid pICHu_(γ)-1M was used asthe source of the heavy chain constant region and hinge region. PlasmidpICHu_(γ)-1M comprises the approximately 6 kb Hind III-Xho I fragment ofa human gamma-1 C gene subcloned into pUC19 that had been linearized bydigestion with Hind III and Sal I. Plasmid pICHu_(γ)-1M was digestedwith Hind III and Eco RI to isolate the 6 kb fragment encoding the humanheavy chain constant region. Plasmid pIC19H was linearized by digestionwith Eco RI. The 1.65 kb PDGFβ-R fragment, the 6 kb heavy chain constantregion fragment and the linearized pIC19H were joined in a three partligation. The resultant plasmid, pSDL111, was digested with Bam HI toisolate the 7.7 kb fragment. Plasmid pμPRE8 was linearized with Bgl IIand was treated with calf intestinal phosphatase to preventself-ligation. The 7.7 kb fragment and the linearized pμPRE8 were joinedby ligation. A plasmid containing the insert in the proper orientationwas designated pSDL113 (FIG. 8).

[0179] Plasmid pSDL113 is linearized by digestion with Cla I and iscotransfected with Pvu I-digested p416 into SP2/0-Ag14 byelectroporation. Transfectants are selected in growth medium containingmethotrexate.

[0180] Media from drug resistant clones are tested for the presence ofsecreted PDGFβ-R analogs by immunoprecipitation using the methoddescribed in Example 12.B.

[0181] E. Cotransfection of pSDL113 with an Immunoglobulin Light ChainGene

[0182] Plasmid pSDL113 is linearized by digestion with Cla I and wascotransfected with pICφ5V_(κ)HuC_(κ)-Neo, which encodes a neomycinresistance gene and a mouse/human chimeric immunoglobulin light chaingene. The mouse immunoglobulin light chain gene was isolated from alambda genomic DNA library constructed from the murine hybridoma cellline NR-ML-05 (Woodhouse et al., ibid.) using an oligonucleotide probedesigned to span the V_(κ)/J_(κ) junction (5′ ACC GAA CGT GAG AGG AGTGCT ATA A 3′; Sequence ID Number 34). The human immunoglobulin lightchain constant region gene was isolated as described in Example 12.B.The mouse NR-ML-05 immunoglobulin light chain variable region gene wassubcloned from the original mouse genomic phage clone into pIC19R as a 3kb Xba I-Hinc II fragment. The human kappa C gene was subcloned from theoriginal human genomic phage clone into pUC19 as a 2.0 kb Hind III-EcoRI fragment. The chimeric kappa gene was created by joining the NR-ML-05light chain variable region gene and human light chain constant regiongene via the common Sph I site and incorporating them with the E. colineomycin resistance gene into pIC19H to yield pICφ5V_(κ)HuC_(κ)-Neo(FIG. 9).

[0183] The linearized pSDL113 and pICφ5V_(κ)HuC_(κ)-Neo are transfectedinto SP2/0-Ag14 cells by electroporation. The transfectants are selectedin growth medium containing methotrexate and neomycin.

[0184] F. Cotransfection of pSDL113 and pSDL114

[0185] A clone of SP2/0-Ag14 stably transfected with pSDL114 and p416was co-transfected with Cla I-digested pSDL113 and Bam HI-digestedpICneo by electroporation. (Plasmid pICneo comprises the SV40 promoteroperatively linked to the E. coli neomycin resistance gene and pIC19Hvector sequences.) Transfected cells were selected in growth mediumcontaining methotrexate and G418. Media from drug-resistant clones weretested for their ability to bind PDGF-BB or PDGF-AA in a competitionbinding assay as described in Example 12.C. The results of the assayshowed that the transfectants secreted a PDGF β-receptor analog whichwas capable of competitively binding PDGF-BB but did not detectably bindto PDGF-AA.

[0186] G. Cotransfection of pSDL114 with Fab

[0187] A clone of SP2/0-AG14 stably transfected with pSDL114 and p416was transfected with the Fab region of the human gamma-4 gene (γ₄) inplasmid pφ5V_(H)Fab-neo.

[0188] Plasmid pφ5V_(H)Fab-neo was constructed by first digestingplasmid p24BRH (Ellison et al., DNA 1: 11, 1988) was digested with Xma Iand Eco RI to isolate the 0.2 kb fragment comprising the immunoglobulin3′ untranslated region. Synthetic oligonucleotides ZC871 (Sequence IDNumber 3; Table 1) and ZC872 (Sequence ID Number 4; Table 1) werekinased and annealed using essentially the methods described by Maniatiset al. (ibid.). The annealed oligonucleotides ZC871/ZC872 formed an SetI-Xma I adapter. The ZC871/ZC872 adapter, the 0.2 kb p24BRH fragment andSst I-Eco RI linearized pUC19 were joined in a three-part ligation toform plasmid pγ₄3′. Plasmid pγ₄3′ was linearized by digestion with BamHI and Hind III. Plasmid p24BRH was cut with Hind III and Bgl II toisolate the 0.85 kb fragment comprising the C_(H)1 region. The pγ₄3′fragment and the Hind III-Bgl II p24BRH fragment were joined by ligationto form plasmid pγ₄Fab. Plasmid pγ₄Fab was digested with Hind III andEco RI to isolate the 1.2 kb fragment comprising γ₄Fab. Plasmid pICneo,comprising the SV40 promoter operatively linked to the E. coli neomycinresistance gene and pIC19H vector sequences, was linearized by digestionwith Sst I and Eco RI. Plasmid pφ5V_(H), comprising the mouseimmunoglobulin heavy chain gene variable region and pUC18 vectorsequences, was digested with Sst I and Hind III to isolate the 5.3 kbV_(H) fragment. The linearized pICneo was joined with the 5.3 kb SstI-Hind III fragment and the 1.2 kb Hind III-Eco RI fragment in athree-part ligation. The resultant plasmid was designatedpφ5V_(H)Fab-neo (FIG. 10).

[0189] A pSDL114/p416-transfected SP2/0-AG14 clone was transfected withSca I-linearized pφ5V_(H)Fab-neo. Transfected cells were selected ingrowth medium containing methotrexate and G418. Media fromdrug-resistant clones were tested for their ability to bind PDGF in acompetition binding assay as described in Example 12.C. The results ofthe assay showed that the PDGF β-receptor analog secreted from thetransfectants was capable of competitively binding PDGF-BB.

[0190] H. Cotransfection of pSDL114 with Fab′

[0191] A stably transfected SP2/0-AG14 isolate containing pSDL114 andp416 was transfected with plasmid pWKI, which contained the Fab′ portionof an immunoglobulin heavy chain gene. Plasmid pWKI was constructed asfollows.

[0192] The immunoglobulin gamma-1 Fab′ sequence, comprising the C_(H)1and hinge regions sequences, was derived from the gamma-1 gene clonedescribed in Example 12.C. The gamma-1 gene clone was digested with HindIII and Eco RI to isolate the 3.0 kb fragment, which was subcloned intoHind III-Eco RI linearized M13mp19. Single-stranded template DNA fromthe resultant phage was subjected to site-directed mutagenesis usingoligonucleotide ZC1447 (Sequence ID Number 9; Table 1) and essentiallythe method of Zoller and Smith (ibid.). A phage clone was identifiedhaving a ZC1447 induced deletion resulting in the fusion of the hingeregion to a DNA sequence encoding the amino acidsAla-Leu-His-Asn-His-Tyr-Thr-Glu-Lys-Ser-Leu-Ser-Leu-Ser-Pro-Gly-Lys(Sequence ID Number 31) followed in-frame by a stop codon. Replicativeform DNA from a positive phage clone was digested with Hind III and EcoRI to isolate the 1.9 kb fragment comprising the C_(H)1 and hingeregions. Plasmid pφ5V_(H) was digested with Sst I and Hind III toisolate the 5.3 kb fragment comprising the mouse immunoglobulin heavychain gene variable region. Plasmid picneo was linearized by digestionwith Sst I and Eco RI. The linearized picneo was joined with the 5.3 kbHind III-Sst I fragment and the 1.9 kb Hind III-Eco RI fragment in athree-part ligation. The resultant plasmid was designated pWKI (FIG.10).

[0193] An SP2/0-AG14 clone stably transfected with pSDL114 and p416 wastransfected with Asp 718-linearized pWKI. Transfected cells wereselected by growth in medium containing methotrexate and G418. Mediasamples from transfected cells were assayed using the competition assaydescribed in Example 12.C. Results from the assays showed that thetransfected cells produced a PDGF β-receptor analog capable ofcompetitively binding PDGF-BB.

Example 13 Purification and Characterization of PDGF β-Receptor Analogsfrom Mammalian Cells Co-transfected with pSDL113 and pSDL114

[0194] A. Purification of PDGF β-Receptor Analogs

[0195] The PDGF β-receptor analog was purified from conditioned culturemedia from a clone of transfected cells grown in a hollow fiber system.The media was passed over a protein-A sepharose column, and the columnwas washed sequentially with phosphate buffered saline, pH 7.2 (PBS;Sigma, St. Louis, Mo.) and 0.1 M citrate, pH 5.0. The PDGF β-receptoranalog was eluted from the protein-A column with 0.1 M citrate pH 2.5and immediately neutralized by the addition of Tris-base, pH 7.4. Theeluate fractions containing PDGF β-receptor analog, as determined bysilver stain, were pooled and chromatographed over an S-200 column(Pharmacia LKB Technologies, Inc., Piscataway, N.J.) equilibrated withPBS. The peak fractions from the S-200 column were pooled andconcentrated on a centriprep-10 concentrator (Amicon). Glycerol (10%final volume) was added to the preparation and the sample frozen at −80°C. PDGF β-receptor analogs purified from pSDL114+pSDL113 co-transfectedcells were termed “tetrameric PDGF α-receptors”.

[0196] B. Measurement of The Relative Binding Affinity of TetramericPDGF β-Receptor Analog by Soluble Receptor Assay

[0197] Purified tetrameric PDGF β-receptor analog was compared todetergent solubilized extracts of human dermal fibroblasts for¹²⁵I-labeled PDGF-BB binding activity in a soluble receptor assayessentially as described by Hart et al. (J. Biol. Chem. 262:10780-10785, 1987). Human dermal fibroblast cells were extracted at20×10⁶ cell equivalents per ml in TNEN extraction buffer (20 mMTris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF,10% glycerol). Two hundred and fifty thousand PDGF β-receptor-subunitsper cell was used to calculate the tetrameric PDGF β-receptor analognumber per volume of extract. This value has been previously publishedby Seifert et al. (J. Biol Chem. 264: 8771-8778, 1989). The PDGFβ-receptor analog number was determined from the protein concentrationof the PDGF β-receptor analog assuming an average molecular weight of140 kDa for each immunoglobulin-PDGF β-receptor monomer, and fourmonomers per tetramer. Thus, each tetrameric molecule contains fourreceptor molecules.

[0198] Increasing amounts of either detergent solubilized extracts ofhuman dermal fibroblast cells or purified PDGF β-receptor analog wereincubated with 1 ng of ¹²⁵I-labeled PDGF-BB for one hour at 37° C. Thesample was then diluted with 1 ml binding media and was added tomonolayers of human dermal fibroblast cells grown in 24-well culturedishes. The samples were incubated for two hours at 4° C. The wells werewashed to remove unbound, ¹²⁵I-labeled PDGF-BB. On half of a milliliterof extraction buffer (PBS+1% Nonidet P-40) was added to each wellfollowed by a 5 minute incubation. The extraction mixtures wereharvested and counted in a gamma counter.

[0199] The results showed that the PDGF β-receptor analog had the samerelative binding affinity as solubilized PDGF β-receptor-subunit frommammalian cells in a solution phase binding assay.

[0200] C. Determination of the Binding Affinity of the PDGF β-ReceptorAnalog in a Solid Phase Format

[0201] The apparent dissociation constant K_(D)(app) of the PDGFβ-receptor analog was determined essentially as described by Bowen-Popeand Ross (Methods in Enzymology 109: 69-100, 1985), using theconcentration of ¹²⁵I-labeled PDGF-BB giving half-maximal specific¹²⁵I-labeled PDGF-BB binding. Saturation binding assays to determine theconcentration of ¹²⁵I-labeled PDGF-BB that gave half-maximal binding toimmobilized PDGF β-receptor analog were conducted as follows.

[0202] Affinity purified goat anti-human IgG, R- and L-chain(Commercially available from Cappel Labs) was diluted into 0.1 MNa₂HCO₃, pH 9.6 to a concentration of 2 μg/ml. One hundred microlitersof the antibody solution was coated onto each well of 96-well microtiterplates for 18 hours at 4° C. The wells were washed once with ELISA Cbuffer (PBS+0.05% Tween-20) followed by an incubation with 175 μl/wellof ELISA B buffer (PBS+1% BSA+0.05% Tween-20) to block the wells. Thewells were washed once with ELISA B buffer. One hundred microliters of12.1 ng/ml or 24.3 ng/ml of tetrameric PDGF β-receptor analog proteindiluted in ELISA B was added to each well and the plates were incubatedfor 2 hours at 37° C. Unbound protein was removed from the wells by twowashes with ELISA C. ¹²⁵I-labeled PDGF-BB_(Tyr) (Example 18.F.) wasserially diluted into binding media (25 mM HEPES, pH 7.2, 0.25% rabbitserum albumin diluted in HAMs F-12 medium (GIBCO-BRL)), and 100 μl ofthe dilutions were added to the wells. The plates were incubated for twohours at room temperature. The unbound ¹²⁵I-labeled PDGF-BB was removed,and the wells were washed three times with binding media. Following thelast wash, 100 μl of 0.1 M citrate, pH 2.5 was added to each well. Afterfive minutes, the citrate buffer was removed, transferred to a tube andcounted in a gamma counter. The counts reflect counts of ¹²⁵I-labeledPDGF-BB_(Tyr) bound by the receptor analog. Nonspecific binding for eachconcentration of ¹²⁵I-labeled PDGF-BB_(Tyr) was determined by a parallelassay wherein separate wells coated only with goat anti-human IgG wereincubated with the ¹²⁵I-labeled PDGF-BB concentrations. Nonspecificbinding was determined to be 2.8% of the total input counts per well andaveraged 6% of the total counts bound.

[0203] Saturation binding assay on 12.1 and 24.3 ng/ml of tetramericPDGF β-receptor analog gave half-maximal binding at 0.8 and 0.82 ng/ml¹²⁵I-labeled PDGF-BB_(Tyr), respectively. By Scatchard analysis(Scatchard, Ann. NY Acad. Sci 51: 660-667, 1949) these values were shownto correspond to a K_(D)(app) of 2.7×10⁻¹¹ which agree with thepublished values for PDGF receptors on mammalian cells.

Example 14 Solid Phase Ligand Binding Assay Using the PDGF β-ReceptorAnalog

[0204] A. Solid Phase Radioreceptor Competition Binding Assay

[0205] In a solid phase radioreceptor competition binding assay (RRA),the wells of 96-well microtiter plates were coated with 100 μl of 2μg/ml affinity purified goat anti-human IgG (Cappel Labs) diluted in 0.1M Na₂HCO₃, pH 9.6. After an eighteen hour incubation at 4° C., the wellswere washed once with ELISA C. The wells were blocked by incubation for2 hours at 37° C. with 175 μl/well ELISA B. The wells were washed oncewith ELISA B then incubated for 2 hours at 37° C. with 50 ng/mltetrameric PDGF β-receptor analog diluted in ELISA B. The unboundreceptor was removed, and the test wells were incubated with increasingconcentrations of serially diluted, unlabeled PDGF-BB (diluted inbinding media. Following a two hour incubation at room temperature, thewells were washed three times with binding media. One hundredmicroliters of 5 ng/ml ¹²⁵I-labeled PDGF-BB_(Tyr) (Example 18.F.) wasadded to each well, and the plates were incubated for an additional twohours at room temperature. The wells were washed three times withbinding media followed by a 5 minute incubation with 100 μl/well of 0.1M citrate, pH 2.5. The samples were harvested and counted in a gammacounter.

[0206] Radioreceptor assay (RRA) competition binding curves weregenerated for PDGF β-receptor analog protein plated at 48.6 ng/ml. Thesensitivity of the assays is 1 ng/ml of PDGF-BB, with 8 ng/ml giving 50%inhibition in ¹²⁵I-PDGF-BB binding, and a working range between 1 and 32ng/ml of PDGF-BB. The values were similar to those obtained usingmonolayers of SK-5 cells in an RRA.

[0207] B. Use of Tetrameric PDGF β-Receptor Analogs As Antagonists forPDGF-Stimulated Mitogenesis.

[0208] A tetrameric PDGF β-receptor analog, purified as described inExample 13, was analyzed for the ability to neutralize PDGF-stimulatedmitogenesis in mouse 3T3 cells. Increasing amounts of the purifiedtetrameric PDGF β-receptor analog were mixed with 5 ng of PDGF. Themixtures were then added to cultures of mouse 3T3 cells. The ability ofthe PDGF to stimulate a mitogenic response, as measured by theincorporation of ³H-thymidine, was determined essentially as described(Raines and Ross, Methods in Enzymology 109: 749-773, 1985, which isincorporated by reference herein). The tetrameric PDGF β-receptor analogdemonstrated a dose response inhibition of PDGF-BB-stimulated³H-thymidine incorporation, while having essentially no effect onPDGF-AA- and PDGF-AB-stimulated ³H-thymidine incorporation.

[0209] C. Binding of Tetrameric PDGF β-receptor Analog to ImmobilizedPDGF.

[0210] A tetrameric PDGF β-receptor analog, purified as described inExample 13, was analyzed for its ability to bind to immobilized PDGF.PDGF-BB (100 ng/ml) was coated onto wells a 96-well microtiter plate,and the plates were incubated 18 hours at 4° C. followed by one washwith ELISA C buffer. The wells were incubated for 2 hours 37° C. withELISA B buffer to block the wells. Increasing concentrations of¹²⁵I-labeled tetrameric PDGF β-receptor analog, diluted in bindingmedia, was added to the wells for two hours at room temperature. Thewells were washed four times with ELISA C buffer to remove unboundreceptor analog. One hundred microliters of 1 M H₂SO₄ was added to eachwell and the plates were incubated for five minutes at room temperature.The solution was then harvested and transferred to tubes to be countedin a gamma counter. Nonspecific binding was determined to be less than10% of the total counts bound.

[0211] A receptor competition binding assay was developed using thisassay format. The assay was carried out as described above, andsimultaneous to the addition of the ¹²⁵I-labeled tetrameric PDGFβ-receptor analog, increasing amounts of PDGF-AA, AB or BB were added tothe PDGF-BB coated wells. Under these condtions, only PDGF-BB was foundto significantly block the binding of the labeled PDGF β-receptor analogto the immobilized PDGF-BB.

Example 15 Construction and Expression of PDGFα-R Analogs in CulturedMouse Myeloma Cells

[0212] A. Construction of an Optimized PDGFα-R cDNA

[0213] The PDGF α-receptor coding region was optimized for expression inmammalian cells as follows. The 5′ end of the cDNA was modified toinclude an optimized Kozak consensus translation initiation sequence(Kozak, Nuc, Acids Res. 12: 857-872, 1984) and Eco RI and Bam HI sitesjust 5′ of the initiation methionine codon. Oligonucleotides ZC2181,ZC2182, ZC2183 and ZC2184 (Sequence ID Numbers 23, 24, 25 and 26,respectively; Table 1) were designed to form, when annealed, an adapterhaving an Eco RI adhesive end, a Bam HI restriction site, a sequenceencoding a Kozak consensus sequence 5′ to the initiating methioninecodon, a mammalian codon optimized sequence encoding amino acids 1-42 ofFIG. 11, and an Eco RI adhesive end that destroys the Eco RI site withinthe PDGFα-R coding sequence. The adapter also introduced a diagnosticCla I site 3′ to the initiation methionine codon. OligonucleotidesZC2181, ZC2182, ZC2183 and ZC2184 were kinased, annealed and ligated.Plasmid pα17B was linearized by partial digestion with Eco RI. Thelinearized pα17B was ligated with the ZC2181/ZC2182/ZC2183/ZC2184oligonucleotide adapter, and the ligation mixture was transformed intoE. coli Plasmid DNA prepared from the transformants was analyzed byrestriction analysis and a positive clone having the oligonucleotideadapter in the correct orientation was digested with Eco RI and Pst I toisolate the 1.6 kb fragment. This fragment was subcloned into EcoRI+PstI-linearized M13mp19. The resultant phage clone was designated 792-8.Single-stranded 792-8 DNA was sequenced to confirm the orientation ofthe adapter.

[0214] A fragment encoding the ligand-binding domain of the PDGFα-receptor (PDGFα-R) was then generated as follows. Restriction sitesand a splice donor sequence were introduced at the 3′ end of the PDGFα-Rextracellular domain by PCR amplification of the 792-8 DNA andoligonucleotides ZC2311 and ZC2392 (Sequence ID Numbers 27 and 30, Table1). Oligonucleotide ZC2311 is a sense primer encoding nucleotides 1470to 1489 of FIG. 11. Oligonucleotide ZC2392 is an antisense primer thatencodes nucleotides 1759 to 1776 of FIG. 11 followed by a splice donorand Xba I and Hind III restriction sites. The 792-8 DNA was amplifiedusing manufacturer recommended (Perkin Elmer Cetus, Norwalk, Conn.)conditions and the GeneAmp™ DNA amplification reagent kit (Perkin ElmerCetus), and blunt-ended 329 bp fragment was isolated. The blunt-endfragment was digested with Nco I and Hind III and ligated with SmaI-digested pUC18. A plasmid having an insert with the Nco I site distalto the Hind III site present in the pUC18 polylinker was designatedpUC18 Sma-PCR Nco HIII #13. The Hind III site present in the insert wasnot regenerated upon ligation with the linearized pUC18. Plasmid pUC18Sma-PCR Nco HIII #13 was digested with Nco I and Hind III to isolate the355 bp PDGFα-R containing fragment encoding PDGFαR. OligonucleotidesZC2351 and ZC2352 (Table 1; Sequence ID Numbers 28 and 29) were kinasedand annealed to form an Sst I-Nco I adapter encoding an internal Eco RIsite and a Kozak consensus translation initiation site. The 355 bp NcoI-Hind III fragment, the ZC2351/ZC2352 adapter and a 1273 bp Nco Ifragment comprising the extracellular domain of of PDGF α-R derived from792-8 were ligated with Hind III+SstI-digested pUC18 and tranformed intoE. coli. Plasmid DNA was isolated from the transformants and analyzed byrestriction analysis. None of the isolates contained the 1273 bp Nco Ifragment. A plasmid containing the Nco I-Hind III fragment and theZC2351/ZC2352 adapter was desginated pUC18 Hin Sst Δ Nco #46. PlasmidpUC18 Hin Sst ΔNco #46 was linearized by digestion and joined byligation with the 1273 bp Nco I fragment comprising the extracellulardomain of the PDGFα-R from clone α18 R-19. The ligations weretransformed into E. coli, and plasmid DNA was isolated from thetransformants. Analysis of the plasmid DNA showed that only clones withthe Nco I fragment in the wrong orientation were isolated. A clonehaving the Nco I fragment in the wrong orientation was digested with NcoI, religated and transformed into E. coli. Plasmid DNA was isolated fromthe transformants and was analyzed by restriction analysis. A plasmidhaving the Nco I insert in the correct orientation was digested tocompletion with Hind III and partially digested with Sst I to isolatethe 1.6 kb fragment comprising the extracellular domain of the PDGFα-Rpreceded by a consensus initiation sequence (Kozak, ibid.) and followedby a splice donor site.

[0215] B. Construction of pPAB7

[0216] The DNA sequence encoding the extracellular domain of the PDGFα-Rwas joined to the immunoglobulin μ enhancer-promoter and to a DNAsequence encoding an immunoglobulin light chain constant region. Theimmunoglobulin μ enhancer-promoter was obtained from plasmid pJH1 whichwas derived from plasmid PICμPRE1 (Example 12.A.) by digestion with EcoRI and Sst I to isolate the 2.2 kb fragment comprising theimmunoglobulin enhancer and heavy chain variable region promoter. The2.2 kb Sst I-Eco RI fragment was ligated with Sst I+Eco RI-linearizedpUC19. The resulting plasmid, designated pJH1, contained theimmunoglobulin enhancer and heavy chain variable region promoterimmediately 5′ to the pUC19 linker sequences. Plasmid pH1 was linearizedby digestion with Sst I and Hind III and joined with the 1.6 kb partialSst I-Hind III fragment containing the PDGFα-R extracellular domainsequences. The resulting plasmid having the immunoglobulin μenhancer-promoter joined to the PDGFα-R extracellular domain wasdesignated pPAB6. Plasmid pSDL112 was digested with Hind III to isolatethe 1.2 kb fragment encoding the immunoglobulin light chain constantregion (Cκ). The 1.2 kb Hind III fragment was ligated with HindIII-linearized pPAB6. A plasmid having the C_(κ) sequence in the correctorientation was desginated pPAB7.

[0217] C. Construction of pPAB9

[0218] The partial Sst I-Hind III fragment encoding the extracellulardomain of the PDGFα-R was joined to the immunoglobulin heavy chainconstant region. For convenience, the internal Xba I site in plasmidpJH1 was removed by digestion with Xba I, blunt-ending with T4 DNApolymerase, and religation. A plasmid which did not contain the internalXba I site, but retained the Xba I site in the polylinker was desginated11.28.3.6. Plasmid 11.28.3.6 was linearized by digestion with Sst I andXba I. Plasmid pPAB6 was digested to completion with Hind III andpartially digested with Sst I to isolate the 1.6 kb Sst I-Hind IIIfragment containing the PDGFα-R extracellular domain. Plasmidpφ5V_(H)huC_(γ)1M-neo (Example 12.C.) was digested wtih Hind III and XbaI to isolate the 6.0 kb fragment encoding the immunoglobulin heavy chainconstant region (huC_(γ)1M). The Sst I-Hind III-linearized 11.28.3.6,the 1.6 kb Sst I-Hind III PDGFα-R fragment and the 6.0 kb Hind III-Xba IhuC_(γ)1M fragment were ligated to form plasmid pPAB9.

[0219] D. Expression of pPAB9 in Mammalian Cells

[0220] Bg1 II-linearized pPAB7 and Pvu I-linearized pPAB9 werecotransfected with Pvu I-linearized p416 into SP2/0-Ag14 cells byelectroporation. Transfected cells were initially selected in growthmedium containing 50 nM methotrexate and were subsequently amplified ina growth medium containing 100 μM methotrexate. Media from drugresistant clones were tested for the presence of secreted PDGFα-receptor analogs by enzyme-linked immunosorbant assay (ELISA).Ninety-six well assay plates were prepared by incubating 100 μl of 1μg/ml monoclonal antibody 292.1.8 which is specific for the PDGFβ-receptor diluted in phosphate buffered saline (PBS; Sigma] overnightat 4° C. Excess antibody was removed by three washes with 0.5% Tween 20in PBS. One hundred microliters of spent media was added to each well,and the plates were incubated for one hour at 4° C. Unbound proteinswere removed by eight washes with 0.5% Tween 20 in PBS. One hundredmicroliters of peroxidase-conjugated goat anti-human IgG heavy chainantibody (diluted 1:1000 in a solution containing 5% chicken serum(GIBCO-BRL)+0.5% Tween 20 in PBS) was added to each well, and the plateswere incubated for one hour at 4° C. One hundred microliters ofchromophore (100 μl ABTS [2,2′-Azinobis(3-ethylbenz-thiazoline sulfonicacid] diammonium salt; Sigma]+1 μl 30% H₂O₂+12.5 ml citrate/phosphatebuffer [9.04 g/l citric acid, 10.16 g/l Na₂HPO₄]) was added to eachwell, and the wells were incubated for 30 minutes at room temperature.The samples were measured at 405 nm. The results of the assay showedthat the PDGF α-receptor analogs secreted by the transfectants containedan immunoglobulin heavy chain.

[0221] Analysis of spent media from transfected cells by Northernanalysis, Western analysis and by radioimmunoprecipitation showed thatthe transfectants did not express a PDCF α-receptor analog from thepPAB7 construction. Transfectants were subsequently treated ascontaining only pPAB9.

[0222] Drug resistant clones was also tested for the presence ofsecreted PDGF α-receptor analogs by immunoprecipitation. For each clone,approximately one million drug resistant transfectants were grown inDMEM lacking cysteine+2% calf serum for 18 hours at 37° C. in thepresence of 50 μCi ³⁵S-cysteine. The spent media was harvested from thelabeled cells and 250 μl of medium from each clone was assayed forbinding to the anti-PDGF α-receptor antibody 292.18. Monoclonal antibody292.18 diluted in PBS was added to each sample to a final concentrationof 2.5 μg per 250 μl spent media. Five microliters of rabbit anti-mouseIg diluted in PBS was added to each sample, and the immunocomplexes wereprecipitated by the addition of 50 μl 10% fixed Staph A (weight/volumein PBS). The immunocomplexes were analyzed on 8% SDS-polyacrylamide gelsfollowed by autoradiography overnight at −70° C. The results of theimmunoprecipitation showed that the PDGF α-receptor analog secreted bythe transfectants was bound by the anti-PDGF α-receptor antibody. Thecombined results of the ELISA and immunoprecipitation assays showed thatthe PDGF α-receptor analog secreted by the transfectants contained boththe PDGF α-receptor ligand-binding domain and the human heavy chain.

[0223] Spent medium from drug-resistant clones were tested for theirability to bind PDGF in a competition binding assay essentially asdescribed in Example 12.C. The results of the assay showed that thetransfectants secreted a PDGF α-receptor analog capable of bindingPDGF-AA. A clone containing the pPAB9 was desginated 3.17.1.57.

[0224] E. Co-expression of pPAB7 and pPAB9 in Mammalian Cells

[0225] Bgl II-linearized pPAB7 and Bam HI-linearized pICneo werecotransfected into clone 3.17.1.57, and transfected cells were selectedin the presence of neomycin. Media from drug resistant cells wereassayed for the presence of immunoglobulin heavy chain, immunoglobulinlight chain and the PDGF α-receptor ligand-binding domain by ELISAessentially as described above. Briefly, ninety-six well assay plateswere prepared by incubating 100 μl of 1 μg/ml goat anti-human IgG Fcantibody (Sigma) or 100 μl of 1 μg/ml 292.18 overnight at 4° C. Excessantibody was removed by three washes with 0.5% Tween 20 in PBS. Onehundred microliters of spent media was added to each well of each plate,and the plates were incubated for one hour at 4° C. Unbound proteinswere removed by eight washes with 0.5% Tween 20 in PBS. One hundredmicroliters of peroxidase-conjugated goat anti-human IgG antibody(diluted 1:1000 in a solution containing 5% chicken serum(GIBCO-BRL)+0.5% Tween 20 in PBS) was added to each well of the platecoated with the anti-Fc antibody, and 100 μl of peroxidase-conjugatedgoat anti human kappa antibody (diluted 1:1000 in a solution containing5% chicken serum (GIBCO-BRL)+0.5% Tween 20 in PBS) was added to eachwell of the plate coated with 292.18. The plates were incubated for onehour at 4° C. One hundred microliters of chromophore (100 μl ABTS[2,2′-Azinobis(3-ethylbenz-thiazoline sulfonic acid) diammonium salt;Sigma]+1 μl 30% H₂O₂+12.5 ml citrate/phosphate buffer [9.04 g/l citricacid, 10.16 g/l Na₂HPO₄]) was added to each well of each plate, and theplates were incubated to 30 minutes at room temperature. The sampleswere measured at 405 nm, the wavelength giving maximal absorbance of thechromogenic substrate, to identify clones having absorbances higher thanbackground indicating the presence of immunoglobulin heavy chain. Clonesthat gave positive results in both ELISA assays (showing that the clonesproduced proteins containing heavy chain regions, light chain constantregions and the PDGF α-receptor ligand-binding region) were selected forfurther characterization.

[0226] Drug resistant clones that were positive for both ELISA assayswere subsequently tested for the presence of secreted PDGF α-receptoranalogs by immunoprecipitation. For each positive clone, approximatelyone million drug resistant transfectants were grown in DMEM lackingcysteine+2% calf serum for 18 hours at 37° C. in the presence of 50 μCI³⁵S-cysteine. The spent media was harvested from the labeled cells and250 μl of medium from each clone was assayed for binding to monoclonalantibody 292.18. Monoclonal antibody 292.18 diluted in PBS was added toeach sample to a final concentration of 2.5 μg. Five microliters ofrabbit anti-mouse Ig diluted in PBS was added to each sample and theimmunocomplexes were precipitated by the addition of 50 μl 10% fixedStaph A (weight/volume in PBS). The immunocomplexes were analyzed on 8%SDS-polyacrylamide gels followed by autoradiography overnight at −70° C.The results of the immunoprecipitation showed that the PDGF α-receptoranalog secreted by the transfectants was bound by the anti-PDGFα-receptor antibody. The combined results of the ELISA andimmunoprecipitation assays showed that the PDGF α-receptor analogsecreted by the transfectants contained the PDGF α-receptorligand-binding domain, the human heavy chain and the human light chainconstant region. A clone that secreted a PDGF α-receptor analog that waspositive for both the above-described ELISA assays and theimmunoprecipitation assay was designated 5.6.2.1.

Example 16 Purification and Characterization of PDGF α-Receptor Analogs

[0227] A. Purification of PDGF α-Receptor Analogs From Clone 3.17.1.57

[0228] The PDGF α-Receptor analog was purified from the conditionedculture media of clone 3.17.1.57 by cycling cell-conditioned medium overan immunoaffinity column composed of monoclonal antibody 292.18 bound toa CNBr-activated Sepharose 4B resin, which is specific for the PDGFα-receptor. The column was washed with PBA, then eluted with 0.1 Mcitrate, pH 3.0. The peak column fractions containing the α-receptorwere pooled, neutralized to pH 7.2 by the addition of 2 M Tris, pH 7.4,then passed over a protein-A Sepharose column. This column was washedsequentially with PBS, then with 0.1 M citrate, pH 5.0. The PDGFα-receptor analog was then eluted with 0.1 M citrate, pH 3.0. The peakeluate fractions were pooled, and glycerol was added to a finalconcentration of 10%. The sample was concentrated on a centriprep 10concentrator (Amicon). The PDGF α-receptor analog purified from clone3.17.1.57 was termed a “dimeric PDGF α-receptor analog”.

[0229] B. Purification of PDGF α-Receptor Analogs From Clone 5.6.2.1

[0230] The PDGF α-receptor analog was purified from the conditionedculture media of clone 5.6.2.1 by cycling cell-conditioned medium overthe immunoaffinity column described above. The column was washed withPBS then eluted with 0.1 M citrate, pH 3.0. The peak column fractionscontaining the α-receptor were pooled, neutralized to pH 7.2 by theaddition of 2 M Tris (what pH 7.4), then passed over a protein-Asepharose column. This column was washed sequentially with PBS then with0.1 M citrate, pH 5.0. The PDGF α-receptor analog was then eluted with0.M citrate, pH 3.0. The peak eluate fractions were pooled and glycerolwas added to a final concentration of 10%. The sample was concentratedon a centriprep 10 concentrator. The PDGF α-receptor analogs purifiedfrom clone 5.6.2.1 was termed a “tetrameric PDGF α-receptor analog”.

Example 17

[0231] A. Use of the PDGF α-receptor Analogs in Ligand Binding Studies

[0232] Purified tetrameric PDGF α-receptor analog and purified dimericPDGF α-receptor analog were compared to monolayers of a control cellline of canine kidney epithelial cells, which do not naturally expressthe PDGF α-receptor, transfected with the human PDGF α-receptor cDNA forligand binding activity. The dissociation constant (Kd) of the receptorpreparations was determined by saturation binding and subsequentScatchard analysis.

[0233] Ligand binding of the purified PDGF α-receptor analogs wasdetermined using a solid phase binding assay. Affinity-purified goatanti-human IgG was diluted to a concentration of 2 μg/ml in 0.1 MNa₂HCO₃, pH 9.6 and 100 μl/well of the solution was used to coat 96-wellmicrotiter plates for 18 hours at 4° C. Excess antibody was removed fromthe wells with one wash with ELISA C buffer (PBS, 0.05% Tween-20). Theplates were incubated with 175 μl/well of ELISA B buffer (PBS, 1% BSA,0.05% Tween-20) to block the wells, followed by two washes with ELISA Cbuffer. One hundred microliters of 50 ng/ml PDGF α-receptor analog(dimeric or tetrameric) diluted in ELISA buffer B was added to each welland the plates were incubated over night at 4° C. Unbound protein wasremoved from the wells with two washes with ELISA buffer B. ¹²⁵I-labeledPDGF-AA was serially diluted in binding media (Hams F-12, 25 mM HEPES pH7.2, 0.25% rabbit serum albumin), and 100 μl of each dilution was addedto the wells. The samples were incubated for two hours at roomtemperature. Unbound ¹²⁵I-labeled PDGF-AA was removed with three washeswith binding media one hundred microliters of 0.1 M citrate, pH 2.5 wasadded to each well, and the plates were incubated for five minutes.After the incubation, the citrate buffer was removed and transferred toa tube for counting in a gamma counter. Nonspecific binding for eachconcentration of ¹²⁵I-labeled PDGF-AA was determined by a parallel assaywherein separate wells coated only with goat anti-human IgG wereincubated with the ¹²⁵I-labeled PDGF-AA samples.

[0234] A saturation binding assay was performed on alpha T-7 cellstransfected with the PDGF α-receptor. The cells were grown to confluencyin 24-well culture plates. The cells were washed one time with bindingmedia. Iodinated PDGF-AA was serially diluted in binding media. Onemilliliter of each dilution was added to the wells, and the plates wereincubated for 3 hours at 4° C. Unbound ¹²⁵I-labeled PDGF-AA was removedand the cells were washed three times with binding media. PBS containing1% Triton X-100 was added to the cells for 5 minutes. The extracts wereharvested and counted in a gamma counter. Nonspecific binding wasdetermined at a single concentration of ¹²⁵I-labeled PDGF-AA using a500-fold excess PDGF-BB.

[0235] The dissociation constants determined by Scatchard analysis(ibid.) of the saturation binding assays for the tetrameric PDGFα-receptor analog, dimeric PDGF α-receptor analog and the control cells(Table 5). TABLE 5 Dissociation Constants for the Tetrameric PDGFα-Receptor, the Dimeric PDGF α-receptor and control cells Transfectedwith the PDGF α-receptor Receptor kD Tetrameric PDGF α-receptor analog1.6 × 10⁻¹¹ Dimeric PDGF α-receptor analog 8.51 × 10⁻¹¹  Control cells[PDGF α-receptor] 3.7 × 10⁻¹¹

[0236] A solid-phase competition binding assay was established using thetetrameric PDGF α-receptor analog. Ninety six-well microtiter plateswere coated with goat anti-human IgG (2 μg/ml), the wells blocked withELISA B buffer, 50 ng/ml of purified tetrameric PDGF α-receptor analogdiluted in binding media was added, and the plates were incubated twohours at room temperature. Unbound receptor was removed and the wellswere washed with binding media. The plates were incubated for two hoursat room temperature with increasing concentrations of either PDGF-AA orPDGF-BB diluted in binding media. The wells were washed, then incubatedfor two hours at room temperature with 3 ng/ml ¹²⁵I-labeled PDGF-AAdiluted in binding media. Unbound labeled PDGF-AA was removed, the wellswere subsequently washed with binding media, and the bound, labeledPDGF-AA was harvested by the addition of 0.1 M citrate, pH 2.5, asdescribed for the saturation binding studies. PDGF-AB, PDGF-AA andPDGF-BB were found to compete for receptor binding with ¹²⁵I-PDGF-AA.

[0237] B. Use of Tetrameric PDGF α-Receptor Analogs As Antagonists forPDGF-Stimulated Mitogenesis.

[0238] A dimeric PDGF α-receptor analog, purified as described inExample 16.B., was analyzed for the ability to neutralizePDGF-stimulated mitogenesis in mouse 3T3 cells. Increasing amounts ofthe purified tetrameric PDGF α-receptor analog were mixed with PDGF-AA,-AB or -BB ranging 0.6 to 5 ng. The mixtures were then added to culturesof confluent mouse 3T3 cells. The ability of the PDGF to stimulate amitogenic response, as measured by the incorporation of ³-thymidine, wasdetermined essentially as described (Raines and Ross, Methods inEnzymology 109: 749-773, 1985, which is incorporated by referenceherein). The dimeric PDGF α-receptor analog demonstrated a dose responseinhibition of PDGF-stimulated ³H-thymidine incorporation for all threeisoforms of PDGF.

[0239] C. Inverse Ligand-Receptor Radioreceptor Assay

[0240] An inverse ligand-receptor radioreceptor assay was designed toscreen for the presence of PDGF-BB, PDGF-BB binding proteins, PDGF-BBrelated molecules, and PDGF-βreceptor antagonists in test samples.PDGF-BB (100 ng/ml) was coated onto the walls of 96-well microtiterplates, and the plates were incubated at 4° C. for 16 hours. The wellswere washed once with ELISA C buffer and then incubated with ELISA Bbuffer to block the nonspecific binding sites. To the wells were added50 μl of either PDGF standard or a test sample and 50 μl of ¹²⁵I-labeledtetrameric PDGF β-receptor analog. The samples were incubated for onehour at room temperature. The wells were washed once with ELISA Cbuffer, and 0.1 M citrate, pH 2.5 containing 1% NP-40 was added to eachwell to disrupt the ligand-receptor analog bond and elute the boundreceptor analog. The acid wash was collected and counted in a gammacounter. The presence of PDGF or a molecule which mimics PDGF orotherwise interferes with the binding of the well-bound PDGF-BB with itsreceptor will cause a decrease in the binding of the radiolabeledtetrameric PDGF β-receptor. Using this assay, PDGF-BB was found toinhibit receptor binding while PDGF-AA and PDGF-AB caused no significantdecrease in receptor binding.

Example 18 Assay Methods

[0241] A. Preparation of Nitrocellulose Filters for Colony Assay

[0242] Colonies of transformants were tested for secretion of PDGFβ-receptor analogs by first growing the cells on nitrocellulose filtersthat had been laid on top of solid growth medium. Nitrocellulose filters(Schleicher & Schuell, Keene, N.H.) were placed on top of solid growthmedium and were allowed to be completely wetted. Test colonies werepatched onto the wetted filters and were grown at 30° C. forapproximately 40 hours. The filters were then removed from the solidmedium, and the cells were removed by four successive rinses withWestern Transfer Buffer (Table 4). The nitrocellulose filters weresoaked in Western Buffer A (Table 4) for one hour at room temperature ona shaking platform with two changes of buffer. Secreted PDGFβ-R analogswere visualized on the filters described below.

[0243] B. Preparation of Protein Blot Filters

[0244] A nitrocellulose filter was soaked in Western Buffer A (Table 4)without the gelatin and placed in a Minifold (Schleicher & Schuell,Keene, N.H.). Five milliliters of culture supernatant was added withoutdilution to the Minifold wells, and the liquid was allowed to passthrough the nitrocellulose filter by gravity. The nitrocellulose filterwas removed from the minifold and was soaked in Western Buffer A (Table3) for one hour on a shaking platform at room temperature. The bufferwas changed three times during the hour incubation.

[0245] C. Preparation of Western Blot Filters

[0246] The transformants were analyzed by Western blot, essentially asdescribed by Towbin et al. (Proc. Natl. Acad. Sci. USA 76: 4350-4354,1979) and Gordon et al. (U.S. Pat. No. 4,452,901). Culture supernatantsfrom appropriately grown transformants were diluted with three volumesof 95% ethanol. The ethanol mixtures were incubated overnight at −70° C.The precipitates were spun out of solution by centrifugation in an SS-24rotor at 18,000 rpm for 20 minutes. The supernatants were discarded andthe precipitate pellets were resuspended in 200 μl of dH₂O. Two hundredmicroliters of 2×loading buffer (Table 4) was added to each sample, andthe samples were incubated in a boiling water bath for 5 minutes.

[0247] The samples were electrophoresed in a 15% sodium dodecylsulfatepolyacrylamide gel under non-reducing conditions. The proteins wereelectrophoretically transferred to nitrocellulose paper using conditionsdescribed by Towbin et al. (ibid.). The nitrocellulose filters were thenincubated in Western Buffer A (Table 4) for 75 minutes at roomtemperature on a platform rocker.

[0248] D. Processing the Filters for Visualization with Antibody

[0249] Filters prepared as described above were screened for proteinsrecognized by the binding of a PDGF β-receptor specific monoclonalantibody, designated PR7212. The filters were removed from the WesternBuffer A (Table 4) and placed in sealed plastic bags containing a 10 mlsolution comprising 10 μg/ml PR7212 monoclonal antibody diluted inWestern Buffer A. The filters were incubated on a rocking platformovernight at 420 C. or for one hour at room temperature. Excess antibodywas removed with three 15-minute washes with Western Buffer A on ashaking platform at room temperature.

[0250] Ten microliters biotin-conjugated horse anti-mouse antibody(Vector Laboratories, Burlingame, Calif.) in 20 ml Western Buffer A wasadded to the filters. The filters were re-incubated for one hour at roomtemperature on a platform shaker, and unbound conjugated antibody wasremoved with three fifteen-minute washes with Western Buffer A.

[0251] The filters were pre-incubated for one hour at room temperaturewith a solution comprising 50 μl Vectastain Reagent A (VectorLaboratories) in 10 ml of Western Buffer A that had been allowed toincubate at room temperature for 30 minutes before use. The filters werewashed with one quick wash with distilled water followed by three15-minute washes with Western Buffer B (Table 4) at room temperature.The Western Buffer B washes were followed by one wash with distilledwater.

[0252] During the preceding wash step, the substrate reagent wasprepared. Sixty mg of horseradish peroxidase reagent (Bio-Rad, Richmond,Calif.) was dissolved in 20 ml HPLC grade methanol. Ninety millilitersof distilled water was added to the dissolved peroxidase followed by 2.5ml 2 M Tris, pH 7.4 and 3.8 ml 4 M NaCl. One hundred microliters of 30%H₂O₂ was added just before use. The washed filters were incubated with75 ml of substrate and incubated at room temperature for 10 minutes withvigorous shaking. After the 10 minute incubation, the buffer waschanged, and the filters were incubated for an additional 10 minutes.The filters were then washed in distilled water for one hour at roomtemperature. Positives were scored as those samples which exhibitedcoloration.

[0253] E. Processing the Filters For Visualization with anAnti-Substance P Antibody

[0254] Filters prepared as described above were probed with ananti-substance P antibody. The filters were removed from the WesternBuffer A and rinsed with Western transfer buffer, followed by a 5-minutewash in phosphate buffered saline (PBS, Sigma, St. Louis, Mo). Thefilters were incubated with a 10 ml solution containing 0.5 M1-ethyl-3-3-dimethylamino propyl carbodiimide (Sigma) in 1.0 M NH₄Cl for40 minutes at room temperature. After incubation, the filters werewashed three times, for 5 minutes per wash, in PBS. The filters wereblocked with 2% powdered milk diluted in PBS.

[0255] The filters were then incubated with a rat anti-substance Pmonoclonal antibody (Accurate Chemical & Scientific Corp., Westbury,N.Y.). Ten microliters of the antibody was diluted in 10 ml of antibodysolution (PBS containing 20% fetal calf serum and 0.5% Tween-20). Thefilters were incubated at room temperature for 1 hour. Unbound antibodywas removed with four 5-minute washes with PBS.

[0256] The filters were then incubated with a biotin-conjugated rabbitanti-rat peroxidase antibody (Cappel Laboratories, Melvern, Pa.). Theconjugated antibody was diluted 1:1000 in 10 ml of antibody solution for2 hours at room temperature. Excess conjugated antibody was removed withfour 5-minute washes with PBS.

[0257] The filters were pre-incubated for 30 minutes at room temperaturewith a solution containing 50 μl Vectastain Reagent A (VectorLaboratories) and 50 μl Vectastain Reagent B (Vector Laboratories) in 10ml of antibody solution that had been allowed to incubate for 30 minutesbefore use. Excess Vectastain reagents were removed by four 5-minutewashes with PBS.

[0258] During the preceding wash step, the substrate reagent wasprepared. Sixty milligrams of horseradish peroxidase reagent (Bio-RadLaboratories, Richmond, Calif.) was dissolved in 25 ml of HPLC grademethanol. Approximately 100 ml of PBS and 200 μl H₂O₂ were added justbefore use. The filters were incubated with the substrate reagent for 10to 20 minutes. The substrate was removed by a vigorous washing distilledwater.

[0259] F. Iodination of PDGF-BB

[0260] A PDGF-BB mutant molecule having a tyrosine replacing thephenylalanine at position 23 (PDGF-BB_(Tyr)) was iodinated andsubsequently purified, using a purification method which produces125I-labeled PDGF-BB with a higher specific activity thanprimary-labeled material and which was found to substantially decreasethe nonspecific binding component. The PDGF-BB_(Tyr) was labeled usingthe Iodobead method (Pierce Chemical). The labeled protein was gelfiltered over a C-25 desalting column (Pharmacia LKB Technologies)equilibrated with 10 mM acetic acid, 0.25% gelatin and 100 mM NaCl. Thepeak fractions were pooled and pH adjusted to 7.2 by the addition ofTris-base. The labeled mixture was chromatographed over an affinitycolumn composed of PDGF β-receptor analog protein coupled toCnBr-activated Sepharose (Pharmacia LKB Technologies, Inc.). The columnwas washed with phosphate buffered saline and eluted with 0.1 M citrate,pH 2.5 containing 0.25% gelatin. The peak eluate fractions were pooledand assayed by ELISA to determine the PDGF-BA concentration.

[0261] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be evident that certain changes and modificationsmay be practiced within the scope of the appended claims.

1 36 4465 base pairs nucleic acid double linear cDNA N N Homo sapiensAdult Skin fibroblasts pR-rX1 CDS 354..3671 1 CCCTCAGCCC TGCTGCCCAGCACGAGCCTG TGCTCGCCCT GCCCAACGCA GACAGCCAGA 60 CCCAGGGCGG CCCCTCTGGCGGCTCTGCTC CTCCCGAAGG ATGCTTGGGG AGTGAGGCGA 120 AGCTGGGCGC TCCTCTCCCCTACAGCAGCC CCCTTCCTCC ATCCCTCTGT TCTCCTGAGC 180 CTTCAGGAGC CTGCACCAGTCCTGCCTGTC CTTCTACTCA GCTGTTACCC ACTCTGGGAC 240 CAGCAGTCTT TCTGATAACTGGGAGAGGGC AGTAAGGAGG ACTTCCTGGA GGGGGTGACT 300 GTCCAGAGCC TGGAACTGTGCCCACACCAG AAGCCATCAG CAGCAAGGAC ACC ATG 356 Met 1 CGG CTT CCG GGT GCGATG CCA GCT CTG GCC CTC AAA GGC GAG CTG CTG 404 Arg Leu Pro Gly Ala MetPro Ala Leu Ala Leu Lys Gly Glu Leu Leu 5 10 15 TTG CTG TCT CTC CTG TTACTT CTG GAA CCA CAG ATC TCT CAG GGC CTG 452 Leu Leu Ser Leu Leu Leu LeuLeu Glu Pro Gln Ile Ser Gln Gly Leu 20 25 30 GTC GTC ACA CCC CCG GGG CCAGAG CTT GTC CTC AAT GTC TCC AGC ACC 500 Val Val Thr Pro Pro Gly Pro GluLeu Val Leu Asn Val Ser Ser Thr 35 40 45 TTC GTT CTG ACC TGC TCG GGT TCAGCT CCG GTG GTG TGG GAA CGG ATG 548 Phe Val Leu Thr Cys Ser Gly Ser AlaPro Val Val Trp Glu Arg Met 50 55 60 65 TCC CAG GAG CCC CCA CAG GAA ATGGCC AAG GCC CAG GAT GGC ACC TTC 596 Ser Gln Glu Pro Pro Gln Glu Met AlaLys Ala Gln Asp Gly Thr Phe 70 75 80 TCC AGC GTG CTC ACA CTG ACC AAC CTCACT GGG CTA GAC ACG GGA GAA 644 Ser Ser Val Leu Thr Leu Thr Asn Leu ThrGly Leu Asp Thr Gly Glu 85 90 95 TAC TTT TGC ACC CAC AAT GAC TCC CGT GGACTG GAG ACC GAT GAG CGG 692 Tyr Phe Cys Thr His Asn Asp Ser Arg Gly LeuGlu Thr Asp Glu Arg 100 105 110 AAA CGG CTC TAC ATC TTT GTG CCA GAT CCCACC GTG GGC TTC CTC CCT 740 Lys Arg Leu Tyr Ile Phe Val Pro Asp Pro ThrVal Gly Phe Leu Pro 115 120 125 AAT GAT GCC GAG GAA CTA TTC ATC TTT CTCACG GAA ATA ACT GAG ATC 788 Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu ThrGlu Ile Thr Glu Ile 130 135 140 145 ACC ATT CCA TGC CGA GTA ACA GAC CCACAG CTG GTG GTG ACA CTG CAC 836 Thr Ile Pro Cys Arg Val Thr Asp Pro GlnLeu Val Val Thr Leu His 150 155 160 GAG AAG AAA GGG GAC GTT GCA CTG CCTGTC CCC TAT GAT CAC CAA CGT 884 Glu Lys Lys Gly Asp Val Ala Leu Pro ValPro Tyr Asp His Gln Arg 165 170 175 GGC TTT TCT GGT ATC TTT GAG GAC AGAAGC TAC ATC TGC AAA ACC ACC 932 Gly Phe Ser Gly Ile Phe Glu Asp Arg SerTyr Ile Cys Lys Thr Thr 180 185 190 ATT GGG GAC AGG GAG GTG GAT TCT GATGCC TAC TAT GTC TAC AGA CTC 980 Ile Gly Asp Arg Glu Val Asp Ser Asp AlaTyr Tyr Val Tyr Arg Leu 195 200 205 CAG GTG TCA TCC ATC AAC GTC TCT GTGAAC GCA GTG CAG ACT GTG GTC 1028 Gln Val Ser Ser Ile Asn Val Ser Val AsnAla Val Gln Thr Val Val 210 215 220 225 CGC CAG GGT GAG AAC ATC ACC CTCATG TGC ATT GTG ATC GGG AAT GAG 1076 Arg Gln Gly Glu Asn Ile Thr Leu MetCys Ile Val Ile Gly Asn Glu 230 235 240 GTG GTC AAC TTC GAG TGG ACA TACCCC CGC AAA GAA AGT GGG CGG CTG 1124 Val Val Asn Phe Glu Trp Thr Tyr ProArg Lys Glu Ser Gly Arg Leu 245 250 255 GTG GAG CCG GTG ACT GAC TTC CTCTTG GAT ATG CCT TAC CAC ATC CGC 1172 Val Glu Pro Val Thr Asp Phe Leu LeuAsp Met Pro Tyr His Ile Arg 260 265 270 TCC ATC CTG CAC ATC CCC AGT GCCGAG TTA GAA GAC TCG GGG ACC TAC 1220 Ser Ile Leu His Ile Pro Ser Ala GluLeu Glu Asp Ser Gly Thr Tyr 275 280 285 ACC TGC AAT GTG ACG GAG AGT GTGAAT GAC CAT CAG GAT GAA AAG GCC 1268 Thr Cys Asn Val Thr Glu Ser Val AsnAsp His Gln Asp Glu Lys Ala 290 295 300 305 ATC AAC ATC ACC GTG GTT GAGAGC GGC TAC GTG CGG CTC CTG GGA GAG 1316 Ile Asn Ile Thr Val Val Glu SerGly Tyr Val Arg Leu Leu Gly Glu 310 315 320 GTG GGC ACA CTA CAA TTT GCTGAG CTG CAT CGG AGC CGG ACA CTG CAG 1364 Val Gly Thr Leu Gln Phe Ala GluLeu His Arg Ser Arg Thr Leu Gln 325 330 335 GTA GTG TTC GAG GCC TAC CCACCG CCC ACT GTC CTG TGG TTC AAA GAC 1412 Val Val Phe Glu Ala Tyr Pro ProPro Thr Val Leu Trp Phe Lys Asp 340 345 350 AAC CGC ACC CTG GGC GAC TCCAGC GCT GGC GAA ATC GCC CTG TCC ACG 1460 Asn Arg Thr Leu Gly Asp Ser SerAla Gly Glu Ile Ala Leu Ser Thr 355 360 365 CGC AAC GTG TCG GAG ACC CGGTAT GTG TCA GAG CTG ACA CTG GTT CGC 1508 Arg Asn Val Ser Glu Thr Arg TyrVal Ser Glu Leu Thr Leu Val Arg 370 375 380 385 GTG AAG GTG GCA GAG GCTGGC CAC TAC ACC ATG CGG GCC TTC CAT GAG 1556 Val Lys Val Ala Glu Ala GlyHis Tyr Thr Met Arg Ala Phe His Glu 390 395 400 GAT GCT GAG GTC CAG CTCTCC TTC CAG CTA CAG ATC AAT GTC CCT GTC 1604 Asp Ala Glu Val Gln Leu SerPhe Gln Leu Gln Ile Asn Val Pro Val 405 410 415 CGA GTG CTG GAG CTA AGTGAG AGC CAC CCT GAC AGT GGG GAA CAG ACA 1652 Arg Val Leu Glu Leu Ser GluSer His Pro Asp Ser Gly Glu Gln Thr 420 425 430 GTC CGC TGT CGT GGC CGGGGC ATG CCC CAG CCG AAC ATC ATC TGG TCT 1700 Val Arg Cys Arg Gly Arg GlyMet Pro Gln Pro Asn Ile Ile Trp Ser 435 440 445 GCC TGC AGA GAC CTC AAAAGG TGT CCA CGT GAG CTG CCG CCC ACG CTG 1748 Ala Cys Arg Asp Leu Lys ArgCys Pro Arg Glu Leu Pro Pro Thr Leu 450 455 460 465 CTG GGG AAC AGT TCCGAA GAG GAG AGC CAG CTG GAG ACT AAC GTG ACG 1796 Leu Gly Asn Ser Ser GluGlu Glu Ser Gln Leu Glu Thr Asn Val Thr 470 475 480 TAC TGG GAG GAG GAGCAG GAG TTT GAG GTG GTG AGC ACA CTG CGT CTG 1844 Tyr Trp Glu Glu Glu GlnGlu Phe Glu Val Val Ser Thr Leu Arg Leu 485 490 495 CAG CAC GTG GAT CGGCCA CTG TCG GTG CGC TGC ACG CTG CGC AAC GCT 1892 Gln His Val Asp Arg ProLeu Ser Val Arg Cys Thr Leu Arg Asn Ala 500 505 510 GTG GGC CAG GAC ACGCAG GAG GTC ATC GTG GTG CCA CAC TCC TTG CCC 1940 Val Gly Gln Asp Thr GlnGlu Val Ile Val Val Pro His Ser Leu Pro 515 520 525 TTT AAG GTG GTG GTGATC TCA GCC ATC CTG GCC CTG GTG GTG CTC ACC 1988 Phe Lys Val Val Val IleSer Ala Ile Leu Ala Leu Val Val Leu Thr 530 535 540 545 ATC ATC TCC CTTATC ATC CTC ATC ATG CTT TGG CAG AAG AAG CCA CGT 2036 Ile Ile Ser Leu IleIle Leu Ile Met Leu Trp Gln Lys Lys Pro Arg 550 555 560 TAC GAG ATC CGATGG AAG GTG ATT GAG TCT GTG AGC TCT GAC GGC CAT 2084 Tyr Glu Ile Arg TrpLys Val Ile Glu Ser Val Ser Ser Asp Gly His 565 570 575 GAG TAC ATC TACGTG GAC CCC ATG CAG CTG CCC TAT GAC TCC ACG TGG 2132 Glu Tyr Ile Tyr ValAsp Pro Met Gln Leu Pro Tyr Asp Ser Thr Trp 580 585 590 GAG CTG CCG CGGGAC CAG CTT GTG CTG GGA CGC ACC CTC GGC TCT GGG 2180 Glu Leu Pro Arg AspGln Leu Val Leu Gly Arg Thr Leu Gly Ser Gly 595 600 605 GCC TTT GGG CAGGTG GTG GAG GCC ACG GCT CAT GGC CTG AGC CAT TCT 2228 Ala Phe Gly Gln ValVal Glu Ala Thr Ala His Gly Leu Ser His Ser 610 615 620 625 CAG GCC ACGATG AAA GTG GCC GTC AAG ATG CTT AAA TCC ACA GCC CGC 2276 Gln Ala Thr MetLys Val Ala Val Lys Met Leu Lys Ser Thr Ala Arg 630 635 640 AGC AGT GAGAAG CAA GCC CTT ATG TCG GAG CTG AAG ATC ATG AGT CAC 2324 Ser Ser Glu LysGln Ala Leu Met Ser Glu Leu Lys Ile Met Ser His 645 650 655 CTT GGG CCCCAC CTG AAC GTG GTC AAC CTG TTG GGG GCC TGC ACC AAA 2372 Leu Gly Pro HisLeu Asn Val Val Asn Leu Leu Gly Ala Cys Thr Lys 660 665 670 GGA GGA CCCATC TAT ATC ATC ACT GAG TAC TGC CGC TAC GGA GAC CTG 2420 Gly Gly Pro IleTyr Ile Ile Thr Glu Tyr Cys Arg Tyr Gly Asp Leu 675 680 685 GTG GAC TACCTG CAC CGC AAC AAA CAC ACC TTC CTG CAG CAC CAC TCC 2468 Val Asp Tyr LeuHis Arg Asn Lys His Thr Phe Leu Gln His His Ser 690 695 700 705 GAC AAGCGC CGC CCG CCC AGC GCG GAG CTC TAC AGC AAT GCT CTG CCC 2516 Asp Lys ArgArg Pro Pro Ser Ala Glu Leu Tyr Ser Asn Ala Leu Pro 710 715 720 GTT GGGCTC CCC CTG CCC AGC CAT GTG TCC TTG ACC GGG GAG AGC GAC 2564 Val Gly LeuPro Leu Pro Ser His Val Ser Leu Thr Gly Glu Ser Asp 725 730 735 GGT GGCTAC ATG GAC ATG AGC AAG GAC GAG TCG GTG GAC TAT GTG CCC 2612 Gly Gly TyrMet Asp Met Ser Lys Asp Glu Ser Val Asp Tyr Val Pro 740 745 750 ATG CTGGAC ATG AAA GGA GAC GTC AAA TAT GCA GAC ATC GAG TCC TCC 2660 Met Leu AspMet Lys Gly Asp Val Lys Tyr Ala Asp Ile Glu Ser Ser 755 760 765 AAC TACATG GCC CCT TAC GAT AAC TAC GTT CCC TCT GCC CCT GAG AGG 2708 Asn Tyr MetAla Pro Tyr Asp Asn Tyr Val Pro Ser Ala Pro Glu Arg 770 775 780 785 ACCTGC CGA GCA ACT TTG ATC AAC GAG TCT CCA GTG CTA AGC TAC ATG 2756 Thr CysArg Ala Thr Leu Ile Asn Glu Ser Pro Val Leu Ser Tyr Met 790 795 800 GACCTC GTG GGC TTC AGC TAC CAG GTG GCC AAT GGC ATG GAG TTT CTG 2804 Asp LeuVal Gly Phe Ser Tyr Gln Val Ala Asn Gly Met Glu Phe Leu 805 810 815 GCCTCC AAG AAC TGC GTC CAC AGA GAC CTG GCG GCT AGG AAC GTG CTC 2852 Ala SerLys Asn Cys Val His Arg Asp Leu Ala Ala Arg Asn Val Leu 820 825 830 ATCTGT GAA GGC AAG CTG GTC AAG ATC TGT GAC TTT GGC CTG GCT CGA 2900 Ile CysGlu Gly Lys Leu Val Lys Ile Cys Asp Phe Gly Leu Ala Arg 835 840 845 GACATC ATG CGG GAC TCG AAT TAC ATC TCC AAA GGC AGC ACC TTT TTG 2948 Asp IleMet Arg Asp Ser Asn Tyr Ile Ser Lys Gly Ser Thr Phe Leu 850 855 860 865CCT TTA AAG TGG ATG GCT CCG GAG AGC ATC TTC AAC AGC CTC TAC ACC 2996 ProLeu Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Ser Leu Tyr Thr 870 875 880ACC CTG AGC GAC GTG TGG TCC TTC GGG ATC CTG CTC TGG GAG ATC TTC 3044 ThrLeu Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Trp Glu Ile Phe 885 890 895ACC TTG GGT GGC ACC CCT TAC CCA GAG CTG CCC ATG AAC GAG CAG TTC 3092 ThrLeu Gly Gly Thr Pro Tyr Pro Glu Leu Pro Met Asn Glu Gln Phe 900 905 910TAC AAT GCC ATC AAA CGG GGT TAC CGC ATG GCC CAG CCT GCC CAT GCC 3140 TyrAsn Ala Ile Lys Arg Gly Tyr Arg Met Ala Gln Pro Ala His Ala 915 920 925TCC GAC GAG ATC TAT GAG ATC ATG CAG AAG TGC TGG GAA GAG AAG TTT 3188 SerAsp Glu Ile Tyr Glu Ile Met Gln Lys Cys Trp Glu Glu Lys Phe 930 935 940945 GAG ATT CGG CCC CCC TTC TCC CAG CTG GTG CTG CTT CTC GAG AGA CTG 3236Glu Ile Arg Pro Pro Phe Ser Gln Leu Val Leu Leu Leu Glu Arg Leu 950 955960 TTG GGC GAA GGT TAC AAA AAG AAG TAC CAG CAG GTG GAT GAG GAG TTT 3284Leu Gly Glu Gly Tyr Lys Lys Lys Tyr Gln Gln Val Asp Glu Glu Phe 965 970975 CTG AGG AGT GAC CAC CCA GCC ATC CTT CGG TCC CAG GCC CGC TTG CCT 3332Leu Arg Ser Asp His Pro Ala Ile Leu Arg Ser Gln Ala Arg Leu Pro 980 985990 GGG TTC CAT GGC CTC CGA TCT CCC CTG GAC ACC AGC TCC GTC CTC TAT 3380Gly Phe His Gly Leu Arg Ser Pro Leu Asp Thr Ser Ser Val Leu Tyr 995 10001005 ACT GCC GTG CAG CCC AAT GAG GGT GAC AAC GAC TAT ATC ATC CCC CTG3428 Thr Ala Val Gln Pro Asn Glu Gly Asp Asn Asp Tyr Ile Ile Pro Leu1010 1015 1020 1025 CCT GAC CCC AAA CCC GAG GTT GCT GAC GAG GGC CCA CTGGAG GGT TCC 3476 Pro Asp Pro Lys Pro Glu Val Ala Asp Glu Gly Pro Leu GluGly Ser 1030 1035 1040 CCC AGC CTA GCC AGC TCC ACC CTG AAT GAA GTC AACACC TCC TCA ACC 3524 Pro Ser Leu Ala Ser Ser Thr Leu Asn Glu Val Asn ThrSer Ser Thr 1045 1050 1055 ATC TCC TGT GAC AGC CCC CTG GAG CCC CAG GACGAA CCA GAG CCA GAG 3572 Ile Ser Cys Asp Ser Pro Leu Glu Pro Gln Asp GluPro Glu Pro Glu 1060 1065 1070 CCC CAG CTT GAG CTC CAG GTG GAG CCG GAGCCA GAG CTG GAA CAG TTG 3620 Pro Gln Leu Glu Leu Gln Val Glu Pro Glu ProGlu Leu Glu Gln Leu 1075 1080 1085 CCG GAT TCG GGG TGC CCT GCG CCT CGGGCG GAA GCA GAG GAT AGC TTC 3668 Pro Asp Ser Gly Cys Pro Ala Pro Arg AlaGlu Ala Glu Asp Ser Phe 1090 1095 1100 1105 CTG TAGGGGGCTG GCCCCTACCCTGCCCTGCCT GAAGCTCCCC CCCTGCCAGC 3721 Leu ACCCAGCATC TCCTGGCCTGGCCTGACCGG GCTTCCTGTC AGCCAGGCTG CCCTTATCAG 3781 CTGTCCCCTT CTGGAAGCTTTCTGCTCCTG ACGTGTTGTG CCCCAAACCC TGGGGCTGGC 3841 TTAGGAGGCA AGAAAACTGCAGGGGCCGTG ACCAGCCCTC TGCCTCCAGG GAGGCCAACT 3901 GACTCTGAGC CAGGGTTCCCCCAGGGAACT CAGTTTTCCC ATATGTAAGA TGGGAAAGTT 3961 AGGCTTGATG ACCCAGAATCTAGGATTCTC TCCCTGGCTG ACACGGTGGG GAGACCGAAT 4021 CCCTCCCTGG GAAGATTCTTGGAGTTACTG AGGTGGTAAA TTAACATTTT TTCTGTTCAG 4081 CCAGCTACCC CTCAAGGAATCATAGCTCTC TCCTCGCACT TTTTATCCAC CCAGGAGCTA 4141 GGGAAGAGAC CCTAGCCTCCCTGGCTGCTG GCTGAGCTAG GGCCTAGCTT GAGCAGTGTT 4201 GCCTCATCCA GAAGAAAGCCAGTCTCCTCC CTATGATGCC AGTCCCTGCG TTCCCTGGCC 4261 CGAGCTGGTC TGGGGCCATTAGGCAGCCTA ATTAATGCTG GAGGCTGAGC CAAGTACAGG 4321 ACACCCCCAG CCTGCAGCCCTTGCCCAGGG CACTTGGAGC ACACGCAGCC ATAGCAAGTG 4381 CCTGTGTCCC TGTCCTTCAGGCCCATCAGT CCTGGGGCTT TTTCTTTATC ACCCTCAGTC 4441 TTAATCCATC CACCAGAGTCTAGA 4465 1106 amino acids amino acid linear protein 2 Met Arg Leu ProGly Ala Met Pro Ala Leu Ala Leu Lys Gly Glu Leu 1 5 10 15 Leu Leu LeuSer Leu Leu Leu Leu Leu Glu Pro Gln Ile Ser Gln Gly 20 25 30 Leu Val ValThr Pro Pro Gly Pro Glu Leu Val Leu Asn Val Ser Ser 35 40 45 Thr Phe ValLeu Thr Cys Ser Gly Ser Ala Pro Val Val Trp Glu Arg 50 55 60 Met Ser GlnGlu Pro Pro Gln Glu Met Ala Lys Ala Gln Asp Gly Thr 65 70 75 80 Phe SerSer Val Leu Thr Leu Thr Asn Leu Thr Gly Leu Asp Thr Gly 85 90 95 Glu TyrPhe Cys Thr His Asn Asp Ser Arg Gly Leu Glu Thr Asp Glu 100 105 110 ArgLys Arg Leu Tyr Ile Phe Val Pro Asp Pro Thr Val Gly Phe Leu 115 120 125Pro Asn Asp Ala Glu Glu Leu Phe Ile Phe Leu Thr Glu Ile Thr Glu 130 135140 Ile Thr Ile Pro Cys Arg Val Thr Asp Pro Gln Leu Val Val Thr Leu 145150 155 160 His Glu Lys Lys Gly Asp Val Ala Leu Pro Val Pro Tyr Asp HisGln 165 170 175 Arg Gly Phe Ser Gly Ile Phe Glu Asp Arg Ser Tyr Ile CysLys Thr 180 185 190 Thr Ile Gly Asp Arg Glu Val Asp Ser Asp Ala Tyr TyrVal Tyr Arg 195 200 205 Leu Gln Val Ser Ser Ile Asn Val Ser Val Asn AlaVal Gln Thr Val 210 215 220 Val Arg Gln Gly Glu Asn Ile Thr Leu Met CysIle Val Ile Gly Asn 225 230 235 240 Glu Val Val Asn Phe Glu Trp Thr TyrPro Arg Lys Glu Ser Gly Arg 245 250 255 Leu Val Glu Pro Val Thr Asp PheLeu Leu Asp Met Pro Tyr His Ile 260 265 270 Arg Ser Ile Leu His Ile ProSer Ala Glu Leu Glu Asp Ser Gly Thr 275 280 285 Tyr Thr Cys Asn Val ThrGlu Ser Val Asn Asp His Gln Asp Glu Lys 290 295 300 Ala Ile Asn Ile ThrVal Val Glu Ser Gly Tyr Val Arg Leu Leu Gly 305 310 315 320 Glu Val GlyThr Leu Gln Phe Ala Glu Leu His Arg Ser Arg Thr Leu 325 330 335 Gln ValVal Phe Glu Ala Tyr Pro Pro Pro Thr Val Leu Trp Phe Lys 340 345 350 AspAsn Arg Thr Leu Gly Asp Ser Ser Ala Gly Glu Ile Ala Leu Ser 355 360 365Thr Arg Asn Val Ser Glu Thr Arg Tyr Val Ser Glu Leu Thr Leu Val 370 375380 Arg Val Lys Val Ala Glu Ala Gly His Tyr Thr Met Arg Ala Phe His 385390 395 400 Glu Asp Ala Glu Val Gln Leu Ser Phe Gln Leu Gln Ile Asn ValPro 405 410 415 Val Arg Val Leu Glu Leu Ser Glu Ser His Pro Asp Ser GlyGlu Gln 420 425 430 Thr Val Arg Cys Arg Gly Arg Gly Met Pro Gln Pro AsnIle Ile Trp 435 440 445 Ser Ala Cys Arg Asp Leu Lys Arg Cys Pro Arg GluLeu Pro Pro Thr 450 455 460 Leu Leu Gly Asn Ser Ser Glu Glu Glu Ser GlnLeu Glu Thr Asn Val 465 470 475 480 Thr Tyr Trp Glu Glu Glu Gln Glu PheGlu Val Val Ser Thr Leu Arg 485 490 495 Leu Gln His Val Asp Arg Pro LeuSer Val Arg Cys Thr Leu Arg Asn 500 505 510 Ala Val Gly Gln Asp Thr GlnGlu Val Ile Val Val Pro His Ser Leu 515 520 525 Pro Phe Lys Val Val ValIle Ser Ala Ile Leu Ala Leu Val Val Leu 530 535 540 Thr Ile Ile Ser LeuIle Ile Leu Ile Met Leu Trp Gln Lys Lys Pro 545 550 555 560 Arg Tyr GluIle Arg Trp Lys Val Ile Glu Ser Val Ser Ser Asp Gly 565 570 575 His GluTyr Ile Tyr Val Asp Pro Met Gln Leu Pro Tyr Asp Ser Thr 580 585 590 TrpGlu Leu Pro Arg Asp Gln Leu Val Leu Gly Arg Thr Leu Gly Ser 595 600 605Gly Ala Phe Gly Gln Val Val Glu Ala Thr Ala His Gly Leu Ser His 610 615620 Ser Gln Ala Thr Met Lys Val Ala Val Lys Met Leu Lys Ser Thr Ala 625630 635 640 Arg Ser Ser Glu Lys Gln Ala Leu Met Ser Glu Leu Lys Ile MetSer 645 650 655 His Leu Gly Pro His Leu Asn Val Val Asn Leu Leu Gly AlaCys Thr 660 665 670 Lys Gly Gly Pro Ile Tyr Ile Ile Thr Glu Tyr Cys ArgTyr Gly Asp 675 680 685 Leu Val Asp Tyr Leu His Arg Asn Lys His Thr PheLeu Gln His His 690 695 700 Ser Asp Lys Arg Arg Pro Pro Ser Ala Glu LeuTyr Ser Asn Ala Leu 705 710 715 720 Pro Val Gly Leu Pro Leu Pro Ser HisVal Ser Leu Thr Gly Glu Ser 725 730 735 Asp Gly Gly Tyr Met Asp Met SerLys Asp Glu Ser Val Asp Tyr Val 740 745 750 Pro Met Leu Asp Met Lys GlyAsp Val Lys Tyr Ala Asp Ile Glu Ser 755 760 765 Ser Asn Tyr Met Ala ProTyr Asp Asn Tyr Val Pro Ser Ala Pro Glu 770 775 780 Arg Thr Cys Arg AlaThr Leu Ile Asn Glu Ser Pro Val Leu Ser Tyr 785 790 795 800 Met Asp LeuVal Gly Phe Ser Tyr Gln Val Ala Asn Gly Met Glu Phe 805 810 815 Leu AlaSer Lys Asn Cys Val His Arg Asp Leu Ala Ala Arg Asn Val 820 825 830 LeuIle Cys Glu Gly Lys Leu Val Lys Ile Cys Asp Phe Gly Leu Ala 835 840 845Arg Asp Ile Met Arg Asp Ser Asn Tyr Ile Ser Lys Gly Ser Thr Phe 850 855860 Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Ser Leu Tyr 865870 875 880 Thr Thr Leu Ser Asp Val Trp Ser Phe Gly Ile Leu Leu Trp GluIle 885 890 895 Phe Thr Leu Gly Gly Thr Pro Tyr Pro Glu Leu Pro Met AsnGlu Gln 900 905 910 Phe Tyr Asn Ala Ile Lys Arg Gly Tyr Arg Met Ala GlnPro Ala His 915 920 925 Ala Ser Asp Glu Ile Tyr Glu Ile Met Gln Lys CysTrp Glu Glu Lys 930 935 940 Phe Glu Ile Arg Pro Pro Phe Ser Gln Leu ValLeu Leu Leu Glu Arg 945 950 955 960 Leu Leu Gly Glu Gly Tyr Lys Lys LysTyr Gln Gln Val Asp Glu Glu 965 970 975 Phe Leu Arg Ser Asp His Pro AlaIle Leu Arg Ser Gln Ala Arg Leu 980 985 990 Pro Gly Phe His Gly Leu ArgSer Pro Leu Asp Thr Ser Ser Val Leu 995 1000 1005 Tyr Thr Ala Val GlnPro Asn Glu Gly Asp Asn Asp Tyr Ile Ile Pro 1010 1015 1020 Leu Pro AspPro Lys Pro Glu Val Ala Asp Glu Gly Pro Leu Glu Gly 1025 1030 1035 1040Ser Pro Ser Leu Ala Ser Ser Thr Leu Asn Glu Val Asn Thr Ser Ser 10451050 1055 Thr Ile Ser Cys Asp Ser Pro Leu Glu Pro Gln Asp Glu Pro GluPro 1060 1065 1070 Glu Pro Gln Leu Glu Leu Gln Val Glu Pro Glu Pro GluLeu Glu Gln 1075 1080 1085 Leu Pro Asp Ser Gly Cys Pro Ala Pro Arg AlaGlu Ala Glu Asp Ser 1090 1095 1100 Phe Leu 1105 57 base pairs nucleicacid single linear Other nucleic acid N N ZC871 3 ATTATACGCT CTCTTCCTCAGGTAAATGAG TGCCAGGGCC GGCAAGCCCC CGCTCCA 57 56 base pairs nucleic acidsingle linear Other nucleic acid N N ZC872 4 CCGGGGAGCG GGGGCTTGCCGGCCCTGGCA CTCATTTACC TGAGGAAGAG AGAGCT 56 45 base pairs nucleic acidsingle linear Other nucleic acid N N ZC904 5 CATGGGCACG TAATCTATAGATTCATCCTT GCTCATATCC ATGTA 45 38 base pairs nucleic acid single linearOther nucleic acid N N ZC906 6 AAGCTGTCCT CTGCTTCAGC CAGAGGTCCT GGGCAGCC38 38 base pairs nucleic acid single linear Other nucleic acid N N ZC9067 AAGCTGTCCT CTGCTTCAGC CAGAGGTCCT GGGCAGCC 38 21 base pairs nucleicacid single linear Other nucleic acid N N ZC1380 8 CATGGTGGAA TTCCTGCTGAT 21 29 base pairs nucleic acid single linear Other nucleic acid N NZC1447 9 TGGTTGTGCA GAGCTGAGGA AGAGATGGA 29 55 base pairs nucleic acidsingle linear Other nucleic acid N N ZC1453 10 AATTCATTAT GTTGTTGCAAGCCTTCTTGT TCCTGCTAGC TGGTTTCGCT GTTAA 55 55 base pairs nucleic acidsingle linear Other nucleic acid N N ZC1454 11 GATCTTAACA GCGAAACCAGCTAGCAGGAA CAAGAAGGCT TGCAACAACA TAATG 55 21 base pairs nucleic acidsingle linear Other nucleic acid N N ZC1478 12 ATCGCGAGCA TGCAGATCTG A21 25 base pairs nucleic acid single linear Other nucleic acid N NZC1479 13 AGCTTCAGAT CTGCATGCTG CCGAT 25 52 base pairs nucleic acidsingle linear Other nucleic acid N N ZC1776 14 AGCTGAGCGC AAATGTTGTGTCGAGTGCCC ACCGTGCCCA GCTTAGAATT CT 52 52 base pairs nucleic acid singlelinear Other nucleic acid N N ZC1777 15 CTAGAGAATT CTAAGCTGGG CACGGTGGGCACTCGACACA ACATTTGCGC TC 52 95 base pairs nucleic acid single linearOther nucleic acid N N ZC1846 16 GATCGGCCAC TGTCGGTGCG CTGCACGCTGCGCAACGCTG TGGGCCAGGA CACGCAGGAG 60 GTCATCGTGG TGCCACACTC CTTGCCCTTTAAGCA 95 95 base pairs nucleic acid single linear Other nucleic acid N NZC1847 17 AGCTTGCTTA AAGGGCAAGG AGTGTGGCAC CACGATGACC TCCTGCGTGTCCTGGCCCAC 60 AGCGTTGCGC AGCGTGCAGC GCACCGACAG TGGCC 95 43 base pairsnucleic acid single linear Other nucleic acid N N ZC1886 18 CCAGTGCCAAGCTTGTCTAG ACTTACCTTT AAAGGGCAAG GAG 43 11 base pairs nucleic acidsingle linear Other nucleic acid N N ZC1892 19 AGCTTGAGCG T 11 11 basepairs nucleic acid single linear Other nucleic acid N N ZC1893 20CTAGACGCTC A 11 47 base pairs nucleic acid single linear Other nucleicacid N N ZC1894 21 AGCTTCCAGT TCTTCGGCCT CATGTCAGTT CTTCGGCCTC ATGTGAT47 47 base pairs nucleic acid single linear Other nucleic acid N NZC1895 22 CTAGATCACA TGAGGCCGAA GAACTGACAT GAGGCCGAAG AACTGGA 47 66 basepairs nucleic acid single linear Other nucleic acid N N ZC2181 23AATTCGGATC CACCATGGGC ACCAGCCACC CGGCGTTCCT GGTGTTAGGC TGCCTGCTGA 60CCGGCC 66 71 base pairs nucleic acid single linear Other nucleic acid NN ZC2182 24 TGAGCCTGAT CCTGTGCCAA CTGAGCCTGC CATCGATCCT GCCAAACGAGAACGAGAAGG 60 TTGTGCAGCT A 71 69 base pairs nucleic acid single linearOther nucleic acid N N ZC2183 25 AATTTAGCTG CACAACCTTC TCGTTCTCGTTTGGCAGGAT CGATGGCAGG CTCAGTTGGC 60 ACAGGATCA 69 68 base pairs nucleicacid single linear Other nucleic acid N N ZC2184 26 GGCTCAGGCCGGTCAGCAGG CAGCCTAACA CCAGGAACGC CGGGTGGCTG GTGCCCATGG 60 TGGATCCG 68 20base pairs nucleic acid single linear Other nucleic acid N N ZC2311 27TGATCACCAT GGCTCAACTG 20 10 base pairs nucleic acid single linear Othernucleic acid N N ZC2351 28 CGAATTCCAC 10 26 base pairs nucleic acidsingle linear Other nucleic acid N N ZC2352 29 ATTATACGCA TGGTGGAATTCGAGCT 26 41 base pairs nucleic acid single linear Other nucleic acid NN ZC2392 30 ACGTAAGCTT GTCTAGACTT ACCTTCAGAA CGCAGGGTGG G 41 17 aminoacids amino acid linear peptide C-terminal pWK1 31 Ala Leu His Asn HisTyr Thr Glu Lys Ser Leu Ser Leu Ser Pro Gly 1 5 10 15 Lys 22 base pairsnucleic acid single linear Other nucleic acid N N 32 TGTGACACTCTCCTGGGAGT TA 22 30 base pairs nucleic acid single linear Other nucleicacid N N 33 GCATAGTAGT TACCATATCC TCTTGCACAG 30 25 base pairs nucleicacid single linear Other nucleic acid N N 34 ACCGAACGTG AGAGGAGTGC TATAA25 4054 base pairs nucleic acid double linear cDNA N N Homo sapiensp-alpha-17B CDS 205..3471 35 GCCCTGGGGA CGGACCGTGG GCGGCGCGCA GCGGCGGGACGCGTTTTGGG GACGTGGTGG 60 CCAGCGCCTT CCTGCAGACC CACAGGGAAG TACTCCCTTTGACCTCCGGG GAGCTGCGAC 120 CAGGTTATAC GTTGCTGGTG GAAAAGTGAC AATTCTAGGAAAAGAGCTAA AAGCCGGATC 180 GGTGACCGAA AGTTTCCCAG AGCT ATG GGG ACT TCC CATCCG GCG TTC CTG 231 Met Gly Thr Ser His Pro Ala Phe Leu 1 5 GTC TTA GGCTGT CTT CTC ACA GGG CTG AGC CTA ATC CTC TGC CAG CTT 279 Val Leu Gly CysLeu Leu Thr Gly Leu Ser Leu Ile Leu Cys Gln Leu 10 15 20 25 TCA TTA CCCTCT ATC CTT CCA AAT GAA AAT GAA AAG GTT GTG CAG CTG 327 Ser Leu Pro SerIle Leu Pro Asn Glu Asn Glu Lys Val Val Gln Leu 30 35 40 AAT TCA TCC TTTTCT CTG AGA TGC TTT GGG GAG AGT GAA GTG AGC TGG 375 Asn Ser Ser Phe SerLeu Arg Cys Phe Gly Glu Ser Glu Val Ser Trp 45 50 55 CAG TAC CCC ATG TCTGAA GAA GAG AGC TCC GAT GTG GAA ATC AGA AAT 423 Gln Tyr Pro Met Ser GluGlu Glu Ser Ser Asp Val Glu Ile Arg Asn 60 65 70 GAA GAA AAC AAC AGC GGCCTT TTT GTG ACG GTC TTG GAA GTG AGC AGT 471 Glu Glu Asn Asn Ser Gly LeuPhe Val Thr Val Leu Glu Val Ser Ser 75 80 85 GCC TCG GCG GCC CAC ACA GGGTTG TAC ACT TGC TAT TAC AAC CAC ACT 519 Ala Ser Ala Ala His Thr Gly LeuTyr Thr Cys Tyr Tyr Asn His Thr 90 95 100 105 CAG ACA GAA GAG AAT GAGCTT GAA GGC AGG CAC ATT TAC ATC TAT GTG 567 Gln Thr Glu Glu Asn Glu LeuGlu Gly Arg His Ile Tyr Ile Tyr Val 110 115 120 CCA GAC CCA GAT GTA GCCTTT GTA CCT CTA GGA ATG ACG GAT TAT TTA 615 Pro Asp Pro Asp Val Ala PheVal Pro Leu Gly Met Thr Asp Tyr Leu 125 130 135 GTC ATC GTG GAG GAT GATGAT TCT GCC ATT ATA CCT TGT CGC ACA ACT 663 Val Ile Val Glu Asp Asp AspSer Ala Ile Ile Pro Cys Arg Thr Thr 140 145 150 GAT CCC GAG ACT CCT GTAACC TTA CAC AAC AGT GAG GGG GTG GTA CCT 711 Asp Pro Glu Thr Pro Val ThrLeu His Asn Ser Glu Gly Val Val Pro 155 160 165 GCC TCC TAC GAC AGC AGACAG GGC TTT AAT GGG ACC TTC ACT GTA GGG 759 Ala Ser Tyr Asp Ser Arg GlnGly Phe Asn Gly Thr Phe Thr Val Gly 170 175 180 185 CCC TAT ATC TGT GAGGCC ACC GTC AAA GGA AAG AAG TTC CAG ACC ATC 807 Pro Tyr Ile Cys Glu AlaThr Val Lys Gly Lys Lys Phe Gln Thr Ile 190 195 200 CCA TTT AAT GTT TATGCT TTA AAA GCA ACA TCA GAG CTG GAT CTA GAA 855 Pro Phe Asn Val Tyr AlaLeu Lys Ala Thr Ser Glu Leu Asp Leu Glu 205 210 215 ATG GAA GCT CTT AAAACC GTG TAT AAG TCA GGG GAA ACG ATT GTG GTC 903 Met Glu Ala Leu Lys ThrVal Tyr Lys Ser Gly Glu Thr Ile Val Val 220 225 230 ACC TGT GCT GTT TTTAAC AAT GAG GTG GTT GAC CTT CAA TGG ACT TAC 951 Thr Cys Ala Val Phe AsnAsn Glu Val Val Asp Leu Gln Trp Thr Tyr 235 240 245 CCT GGA GAA GTG AAAGGC AAA GGC ATC ACA ATA CTG GAA GAA ATC AAA 999 Pro Gly Glu Val Lys GlyLys Gly Ile Thr Ile Leu Glu Glu Ile Lys 250 255 260 265 GTC CCA TCC ATCAAA TTG GTG TAC ACT TTG ACG GTC CCC GAG GCC ACG 1047 Val Pro Ser Ile LysLeu Val Tyr Thr Leu Thr Val Pro Glu Ala Thr 270 275 280 GTG AAA GAC AGTGGA GAT TAC GAA TGT GCT GCC CGC CAG GCT ACC AGG 1095 Val Lys Asp Ser GlyAsp Tyr Glu Cys Ala Ala Arg Gln Ala Thr Arg 285 290 295 GAG GTC AAA GAAATG AAG AAA GTC ACT ATT TCT GTC CAT GAG AAA GGT 1143 Glu Val Lys Glu MetLys Lys Val Thr Ile Ser Val His Glu Lys Gly 300 305 310 TTC ATT GAA ATCAAA CCC ACC TTC AGC CAG TTG GAA GCT GTC AAC CTG 1191 Phe Ile Glu Ile LysPro Thr Phe Ser Gln Leu Glu Ala Val Asn Leu 315 320 325 CAT GAA GTC AAACAT TTT GTT GTA GAG GTG CGG GCC TAC CCA CCT CCC 1239 His Glu Val Lys HisPhe Val Val Glu Val Arg Ala Tyr Pro Pro Pro 330 335 340 345 AGG ATA TCCTGG CTG AAA AAC AAT CTG ACT CTG ATT GAA AAT CTC ACT 1287 Arg Ile Ser TrpLeu Lys Asn Asn Leu Thr Leu Ile Glu Asn Leu Thr 350 355 360 GAG ATC ACCACT GAT GTG GAA AAG ATT CAG GAA ATA AGG TAT CGA AGC 1335 Glu Ile Thr ThrAsp Val Glu Lys Ile Gln Glu Ile Arg Tyr Arg Ser 365 370 375 AAA TTA AAGCTG ATC CGT GCT AAG GAA GAA GAC AGT GGC CAT TAT ACT 1383 Lys Leu Lys LeuIle Arg Ala Lys Glu Glu Asp Ser Gly His Tyr Thr 380 385 390 ATT GTA GCTCAA AAT GAA GAT GCT GTG AAG AGC TAT ACT TTT GAA CTG 1431 Ile Val Ala GlnAsn Glu Asp Ala Val Lys Ser Tyr Thr Phe Glu Leu 395 400 405 TTA ACT CAAGTT CCT TCA TCC ATT CTG GAC TTG GTC GAT GAT CAC CAT 1479 Leu Thr Gln ValPro Ser Ser Ile Leu Asp Leu Val Asp Asp His His 410 415 420 425 GGC TCAACT GGG GGA CAG ACG GTG AGG TGC ACA GCT GAA GGC ACG CCG 1527 Gly Ser ThrGly Gly Gln Thr Val Arg Cys Thr Ala Glu Gly Thr Pro 430 435 440 CTT CCTGAT ATT GAG TGG ATG ATA TGC AAA GAT ATT AAG AAA TGT AAT 1575 Leu Pro AspIle Glu Trp Met Ile Cys Lys Asp Ile Lys Lys Cys Asn 445 450 455 AAT GAAACT TCC TGG ACT ATT TTG GCC AAC AAT GTC TCA AAC ATC ATC 1623 Asn Glu ThrSer Trp Thr Ile Leu Ala Asn Asn Val Ser Asn Ile Ile 460 465 470 ACG GAGATC CAC TCC CGA GAC AGG AGT ACC GTG GAG GGC CGT GTG ACT 1671 Thr Glu IleHis Ser Arg Asp Arg Ser Thr Val Glu Gly Arg Val Thr 475 480 485 TTC GCCAAA GTG GAG GAG ACC ATC GCC GTG CGA TGC CTG GCT AAG AAT 1719 Phe Ala LysVal Glu Glu Thr Ile Ala Val Arg Cys Leu Ala Lys Asn 490 495 500 505 CTCCTT GGA GCT GAG AAC CGA GAG CTG AAG CTG GTG GCT CCC ACC CTG 1767 Leu LeuGly Ala Glu Asn Arg Glu Leu Lys Leu Val Ala Pro Thr Leu 510 515 520 CGTTCT GAA CTC ACG GTG GCT GCT GCA GTC CTG GTG CTG TTG GTG ATT 1815 Arg SerGlu Leu Thr Val Ala Ala Ala Val Leu Val Leu Leu Val Ile 525 530 535 GTGATC ATC TCA CTT ATT GTC CTG GTT GTC ATT TGG AAA CAG AAA CCG 1863 Val IleIle Ser Leu Ile Val Leu Val Val Ile Trp Lys Gln Lys Pro 540 545 550 AGGTAT GAA ATT CGC TGG AGG GTC ATT GAA TCA ATC AGC CCG GAT GGA 1911 Arg TyrGlu Ile Arg Trp Arg Val Ile Glu Ser Ile Ser Pro Asp Gly 555 560 565 CATGAA TAT ATT TAT GTG GAC CCG ATG CAG CTG CCT TAT GAC TCA AGA 1959 His GluTyr Ile Tyr Val Asp Pro Met Gln Leu Pro Tyr Asp Ser Arg 570 575 580 585TGG GAG TTT CCA AGA GAT GGA CTA GTG CTT GGT CGG GTC TTG GGG TCT 2007 TrpGlu Phe Pro Arg Asp Gly Leu Val Leu Gly Arg Val Leu Gly Ser 590 595 600GGA GCG TTT GGG AAG GTG GTT GAA GGA ACA GCC TAT GGA TTA AGC CGG 2055 GlyAla Phe Gly Lys Val Val Glu Gly Thr Ala Tyr Gly Leu Ser Arg 605 610 615TCC CAA CCT GTC ATG AAA GTT GCA GTG AAG ATG CTA AAA CCC ACG GCC 2103 SerGln Pro Val Met Lys Val Ala Val Lys Met Leu Lys Pro Thr Ala 620 625 630AGA TCC AGT GAA AAA CAA GCT CTC ATG TCT GAA CTG AAG ATA ATG ACT 2151 ArgSer Ser Glu Lys Gln Ala Leu Met Ser Glu Leu Lys Ile Met Thr 635 640 645CAC CTG GGG CCA CAT TTG AAC ATT GTA AAC TTG CTG GGA GCC TGC ACC 2199 HisLeu Gly Pro His Leu Asn Ile Val Asn Leu Leu Gly Ala Cys Thr 650 655 660665 AAG TCA GGC CCC ATT TAC ATC ATC ACA GAG TAT TGC TTC TAT GGA GAT 2247Lys Ser Gly Pro Ile Tyr Ile Ile Thr Glu Tyr Cys Phe Tyr Gly Asp 670 675680 TTG GTC AAC TAT TTG CAT AAG AAT AGG GAT AGC TTC CTG AGC CAC CAC 2295Leu Val Asn Tyr Leu His Lys Asn Arg Asp Ser Phe Leu Ser His His 685 690695 CCA GAG AAG CCA AAG AAA GAG CTG GAT ATC TTT GGA TTG AAC CCT GCT 2343Pro Glu Lys Pro Lys Lys Glu Leu Asp Ile Phe Gly Leu Asn Pro Ala 700 705710 GAT GAA AGC ACA CGG AGC TAT GTT ATT TTA TCT TTT GAA AAC AAT GGT 2391Asp Glu Ser Thr Arg Ser Tyr Val Ile Leu Ser Phe Glu Asn Asn Gly 715 720725 GAC TAC ATG GAC ATG AAG CAG GCT GAT ACT ACA CAG TAT GTC CCC ATG 2439Asp Tyr Met Asp Met Lys Gln Ala Asp Thr Thr Gln Tyr Val Pro Met 730 735740 745 CTA GAA AGG AAA GAG GTT TCT AAA TAT TCC GAC ATC CAG AGA TCA CTC2487 Leu Glu Arg Lys Glu Val Ser Lys Tyr Ser Asp Ile Gln Arg Ser Leu 750755 760 TAT GAT CGT CCA GCC TCA TAT AAG AAG AAA TCT ATG TTA GAC TCA GAA2535 Tyr Asp Arg Pro Ala Ser Tyr Lys Lys Lys Ser Met Leu Asp Ser Glu 765770 775 GTC AAA AAC CTC CTT TCA GAT GAT AAC TCA GAA GGC CTT ACT TTA TTG2583 Val Lys Asn Leu Leu Ser Asp Asp Asn Ser Glu Gly Leu Thr Leu Leu 780785 790 GAT TTG TTG AGC TTC ACC TAT CAA GTT GCC CGA GGA ATG GAG TTT TTG2631 Asp Leu Leu Ser Phe Thr Tyr Gln Val Ala Arg Gly Met Glu Phe Leu 795800 805 GCT TCA AAA AAT TGT GTC CAC CGT GAT CTG GCT GCT CGC AAC GTC CTC2679 Ala Ser Lys Asn Cys Val His Arg Asp Leu Ala Ala Arg Asn Val Leu 810815 820 825 CTG GCA CAA GGA AAA ATT GTG AAG ATC TGT GAC TTT GGC CTG GCCAGA 2727 Leu Ala Gln Gly Lys Ile Val Lys Ile Cys Asp Phe Gly Leu Ala Arg830 835 840 GAC ATC ATG CAT GAT TCG AAC TAT GTG TCG AAA GGC AGT ACC TTTCTG 2775 Asp Ile Met His Asp Ser Asn Tyr Val Ser Lys Gly Ser Thr Phe Leu845 850 855 CCC GTG AAG TGG ATG GCT CCT GAG AGC ATC TTT GAC AAC CTC TACACC 2823 Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Asn Leu Tyr Thr860 865 870 ACA CTG AGT GAT GTC TGG TCT TAT GGC ATT CTG CTC TGG GAG ATCTTT 2871 Thr Leu Ser Asp Val Trp Ser Tyr Gly Ile Leu Leu Trp Glu Ile Phe875 880 885 TCC CTT GGT GGC ACC CCT TAC CCC GGC ATG ATG GTG GAT TCT ACTTTC 2919 Ser Leu Gly Gly Thr Pro Tyr Pro Gly Met Met Val Asp Ser Thr Phe890 895 900 905 TAC AAT AAG ATC AAG AGT GGG TAC CGG ATG GCC AAG CCT GACCAC GCT 2967 Tyr Asn Lys Ile Lys Ser Gly Tyr Arg Met Ala Lys Pro Asp HisAla 910 915 920 ACC AGT GAA GTC TAC GAG ATC ATG GTG AAA TGC TGG AAC AGTGAG CCG 3015 Thr Ser Glu Val Tyr Glu Ile Met Val Lys Cys Trp Asn Ser GluPro 925 930 935 GAG AAG AGA CCC TCC TTT TAC CAC CTG AGT GAG ATT GTG GAGAAT CTG 3063 Glu Lys Arg Pro Ser Phe Tyr His Leu Ser Glu Ile Val Glu AsnLeu 940 945 950 CTG CCT GGA CAA TAT AAA AAG AGT TAT GAA AAA ATT CAC CTGGAC TTC 3111 Leu Pro Gly Gln Tyr Lys Lys Ser Tyr Glu Lys Ile His Leu AspPhe 955 960 965 CTG AAG AGT GAC CAT CCT GCT GTG GCA CGC ATG CGT GTG GACTCA GAC 3159 Leu Lys Ser Asp His Pro Ala Val Ala Arg Met Arg Val Asp SerAsp 970 975 980 985 AAT GCA TAC ATT GGT GTC ACC TAC AAA AAC GAG GAA GACAAG CTG AAG 3207 Asn Ala Tyr Ile Gly Val Thr Tyr Lys Asn Glu Glu Asp LysLeu Lys 990 995 1000 GAC TGG GAG GGT GGT CTG GAT GAG CAG AGA CTG AGC GCTGAC AGT GGC 3255 Asp Trp Glu Gly Gly Leu Asp Glu Gln Arg Leu Ser Ala AspSer Gly 1005 1010 1015 TAC ATC ATT CCT CTG CCT GAC ATT GAC CCT GTC CCTGAG GAG GAG GAC 3303 Tyr Ile Ile Pro Leu Pro Asp Ile Asp Pro Val Pro GluGlu Glu Asp 1020 1025 1030 CTG GGC AAG AGG AAC AGA CAC AGC TCG CAG ACCTCT GAA GAG AGT GCC 3351 Leu Gly Lys Arg Asn Arg His Ser Ser Gln Thr SerGlu Glu Ser Ala 1035 1040 1045 ATT GAG ACG GGT TCC AGC AGT TCC ACC TTCATC AAG AGA GAG GAC GAG 3399 Ile Glu Thr Gly Ser Ser Ser Ser Thr Phe IleLys Arg Glu Asp Glu 1050 1055 1060 1065 ACC ATT GAA GAC ATC GAC ATG ATGGAC GAC ATC GGC ATA GAC TCT TCA 3447 Thr Ile Glu Asp Ile Asp Met Met AspAsp Ile Gly Ile Asp Ser Ser 1070 1075 1080 GAC CTG GTG GAA GAC AGC TTCCTG TAACTGGCGG ATTCGAGGGG TTCCTTCCAC 3501 Asp Leu Val Glu Asp Ser PheLeu 1085 TTCTGGGGCC ACCTCTGGAT CCCGTTCAGA AAACCACTTT ATTGCAATGCGGAGGTTGAG 3561 AGGAGGACTT GGTTGATGTT TAAAGAGAAG TTCCCAGCCA AGGGCCTCGGGGAGCGTTCT 3621 AAATATGAAT GAATGGGATA TTTTGAAATG AACTTTGTCA GTGTTGCCTCTTGCAATGCC 3681 TCAGTAGCAT CTCAGTGGTG TGTGAAGTTT GGAGATAGAT GGATAAGGGAATAATAGGCC 3741 ACAGAAGGTG AACTTTGTGC TTCAAGGACA TTGGTGAGAG TCCAACAGACACAATTTATA 3801 CTGCGACAGA ACTTCAGCAT TGTAATTATG TAAATAACTC TAACCAAGGCTGTGTTTAGA 3861 TTGTATTAAC TATCTTCTTT GGACTTCTGA AGAGACCACT CAATCCATCCTGTACTTCCC 3921 TCTTGAAACC TGATGTAGCT GCTGTTGAAC TTTTTAAAGA AGTGCATGAAAAACCATTTT 3981 TGAACCTTAA AAGGTACTGG TACTATAGCA TTTTGCTATC TTTTTTAGTGTTAAAGAGAT 4041 AAAGAATAAT AAG 4054 1089 amino acids amino acid linearprotein 36 Met Gly Thr Ser His Pro Ala Phe Leu Val Leu Gly Cys Leu LeuThr 1 5 10 15 Gly Leu Ser Leu Ile Leu Cys Gln Leu Ser Leu Pro Ser IleLeu Pro 20 25 30 Asn Glu Asn Glu Lys Val Val Gln Leu Asn Ser Ser Phe SerLeu Arg 35 40 45 Cys Phe Gly Glu Ser Glu Val Ser Trp Gln Tyr Pro Met SerGlu Glu 50 55 60 Glu Ser Ser Asp Val Glu Ile Arg Asn Glu Glu Asn Asn SerGly Leu 65 70 75 80 Phe Val Thr Val Leu Glu Val Ser Ser Ala Ser Ala AlaHis Thr Gly 85 90 95 Leu Tyr Thr Cys Tyr Tyr Asn His Thr Gln Thr Glu GluAsn Glu Leu 100 105 110 Glu Gly Arg His Ile Tyr Ile Tyr Val Pro Asp ProAsp Val Ala Phe 115 120 125 Val Pro Leu Gly Met Thr Asp Tyr Leu Val IleVal Glu Asp Asp Asp 130 135 140 Ser Ala Ile Ile Pro Cys Arg Thr Thr AspPro Glu Thr Pro Val Thr 145 150 155 160 Leu His Asn Ser Glu Gly Val ValPro Ala Ser Tyr Asp Ser Arg Gln 165 170 175 Gly Phe Asn Gly Thr Phe ThrVal Gly Pro Tyr Ile Cys Glu Ala Thr 180 185 190 Val Lys Gly Lys Lys PheGln Thr Ile Pro Phe Asn Val Tyr Ala Leu 195 200 205 Lys Ala Thr Ser GluLeu Asp Leu Glu Met Glu Ala Leu Lys Thr Val 210 215 220 Tyr Lys Ser GlyGlu Thr Ile Val Val Thr Cys Ala Val Phe Asn Asn 225 230 235 240 Glu ValVal Asp Leu Gln Trp Thr Tyr Pro Gly Glu Val Lys Gly Lys 245 250 255 GlyIle Thr Ile Leu Glu Glu Ile Lys Val Pro Ser Ile Lys Leu Val 260 265 270Tyr Thr Leu Thr Val Pro Glu Ala Thr Val Lys Asp Ser Gly Asp Tyr 275 280285 Glu Cys Ala Ala Arg Gln Ala Thr Arg Glu Val Lys Glu Met Lys Lys 290295 300 Val Thr Ile Ser Val His Glu Lys Gly Phe Ile Glu Ile Lys Pro Thr305 310 315 320 Phe Ser Gln Leu Glu Ala Val Asn Leu His Glu Val Lys HisPhe Val 325 330 335 Val Glu Val Arg Ala Tyr Pro Pro Pro Arg Ile Ser TrpLeu Lys Asn 340 345 350 Asn Leu Thr Leu Ile Glu Asn Leu Thr Glu Ile ThrThr Asp Val Glu 355 360 365 Lys Ile Gln Glu Ile Arg Tyr Arg Ser Lys LeuLys Leu Ile Arg Ala 370 375 380 Lys Glu Glu Asp Ser Gly His Tyr Thr IleVal Ala Gln Asn Glu Asp 385 390 395 400 Ala Val Lys Ser Tyr Thr Phe GluLeu Leu Thr Gln Val Pro Ser Ser 405 410 415 Ile Leu Asp Leu Val Asp AspHis His Gly Ser Thr Gly Gly Gln Thr 420 425 430 Val Arg Cys Thr Ala GluGly Thr Pro Leu Pro Asp Ile Glu Trp Met 435 440 445 Ile Cys Lys Asp IleLys Lys Cys Asn Asn Glu Thr Ser Trp Thr Ile 450 455 460 Leu Ala Asn AsnVal Ser Asn Ile Ile Thr Glu Ile His Ser Arg Asp 465 470 475 480 Arg SerThr Val Glu Gly Arg Val Thr Phe Ala Lys Val Glu Glu Thr 485 490 495 IleAla Val Arg Cys Leu Ala Lys Asn Leu Leu Gly Ala Glu Asn Arg 500 505 510Glu Leu Lys Leu Val Ala Pro Thr Leu Arg Ser Glu Leu Thr Val Ala 515 520525 Ala Ala Val Leu Val Leu Leu Val Ile Val Ile Ile Ser Leu Ile Val 530535 540 Leu Val Val Ile Trp Lys Gln Lys Pro Arg Tyr Glu Ile Arg Trp Arg545 550 555 560 Val Ile Glu Ser Ile Ser Pro Asp Gly His Glu Tyr Ile TyrVal Asp 565 570 575 Pro Met Gln Leu Pro Tyr Asp Ser Arg Trp Glu Phe ProArg Asp Gly 580 585 590 Leu Val Leu Gly Arg Val Leu Gly Ser Gly Ala PheGly Lys Val Val 595 600 605 Glu Gly Thr Ala Tyr Gly Leu Ser Arg Ser GlnPro Val Met Lys Val 610 615 620 Ala Val Lys Met Leu Lys Pro Thr Ala ArgSer Ser Glu Lys Gln Ala 625 630 635 640 Leu Met Ser Glu Leu Lys Ile MetThr His Leu Gly Pro His Leu Asn 645 650 655 Ile Val Asn Leu Leu Gly AlaCys Thr Lys Ser Gly Pro Ile Tyr Ile 660 665 670 Ile Thr Glu Tyr Cys PheTyr Gly Asp Leu Val Asn Tyr Leu His Lys 675 680 685 Asn Arg Asp Ser PheLeu Ser His His Pro Glu Lys Pro Lys Lys Glu 690 695 700 Leu Asp Ile PheGly Leu Asn Pro Ala Asp Glu Ser Thr Arg Ser Tyr 705 710 715 720 Val IleLeu Ser Phe Glu Asn Asn Gly Asp Tyr Met Asp Met Lys Gln 725 730 735 AlaAsp Thr Thr Gln Tyr Val Pro Met Leu Glu Arg Lys Glu Val Ser 740 745 750Lys Tyr Ser Asp Ile Gln Arg Ser Leu Tyr Asp Arg Pro Ala Ser Tyr 755 760765 Lys Lys Lys Ser Met Leu Asp Ser Glu Val Lys Asn Leu Leu Ser Asp 770775 780 Asp Asn Ser Glu Gly Leu Thr Leu Leu Asp Leu Leu Ser Phe Thr Tyr785 790 795 800 Gln Val Ala Arg Gly Met Glu Phe Leu Ala Ser Lys Asn CysVal His 805 810 815 Arg Asp Leu Ala Ala Arg Asn Val Leu Leu Ala Gln GlyLys Ile Val 820 825 830 Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile MetHis Asp Ser Asn 835 840 845 Tyr Val Ser Lys Gly Ser Thr Phe Leu Pro ValLys Trp Met Ala Pro 850 855 860 Glu Ser Ile Phe Asp Asn Leu Tyr Thr ThrLeu Ser Asp Val Trp Ser 865 870 875 880 Tyr Gly Ile Leu Leu Trp Glu IlePhe Ser Leu Gly Gly Thr Pro Tyr 885 890 895 Pro Gly Met Met Val Asp SerThr Phe Tyr Asn Lys Ile Lys Ser Gly 900 905 910 Tyr Arg Met Ala Lys ProAsp His Ala Thr Ser Glu Val Tyr Glu Ile 915 920 925 Met Val Lys Cys TrpAsn Ser Glu Pro Glu Lys Arg Pro Ser Phe Tyr 930 935 940 His Leu Ser GluIle Val Glu Asn Leu Leu Pro Gly Gln Tyr Lys Lys 945 950 955 960 Ser TyrGlu Lys Ile His Leu Asp Phe Leu Lys Ser Asp His Pro Ala 965 970 975 ValAla Arg Met Arg Val Asp Ser Asp Asn Ala Tyr Ile Gly Val Thr 980 985 990Tyr Lys Asn Glu Glu Asp Lys Leu Lys Asp Trp Glu Gly Gly Leu Asp 995 10001005 Glu Gln Arg Leu Ser Ala Asp Ser Gly Tyr Ile Ile Pro Leu Pro Asp1010 1015 1020 Ile Asp Pro Val Pro Glu Glu Glu Asp Leu Gly Lys Arg AsnArg His 1025 1030 1035 1040 Ser Ser Gln Thr Ser Glu Glu Ser Ala Ile GluThr Gly Ser Ser Ser 1045 1050 1055 Ser Thr Phe Ile Lys Arg Glu Asp GluThr Ile Glu Asp Ile Asp Met 1060 1065 1070 Met Asp Asp Ile Gly Ile AspSer Ser Asp Leu Val Glu Asp Ser Phe 1075 1080 1085 Leu

1. A method for producing a secreted, biologically active dimerizedpolypeptide fusion, comprising: introducing into a eukaryotic host cella DNA construct comprising a transcriptional promoter operatively linkedto a secretory signal sequence followed downstream by and in properreading frame with a DNA sequence encoding a non-immunoglobulinpolypeptide requiring dimerization for biological activity joined to adimerizing protein; growing said host cell in an appropriate growthmedium under physiological conditions to allow the secretion of adimerized polypeptide fusion encoded by said DNA sequence; and isolatingsaid dimerized polypeptide fusion from said host cell.
 2. A method forproducing a secreted, biologically active dimerized polypeptide fusion,comprising: introducing into a eukaryotic host cell a first DNAconstruct comprising a transcriptional promoter operatively linked to afirst secretory signal sequence followed downstream by and in properreading frame with a first DNA sequence encoding a non-immunoglobulinpolypeptide requiring dimerization for biological activity joined to animmunoglobulin light chain constant region; introducing into said hostcell a second DNA construct comprising a transcriptional promoteroperatively linked to a second secretory signal sequence followeddownstream by and in proper reading frame with a second DNA sequenceencoding an immunoglobulin heavy chain constant region domain selectedfrom the group consisting of C_(H)1, C_(H)2, C_(H)3, and C_(H)4; growingsaid host cell in an appropriate growth medium under physiologicalconditions to allow the secretion of a biologically active dimerizedpolypeptide fusion encoded by said first and second DNA sequences; andisolating said biologically active dimerized polypeptide fusion fromsaid host cell.
 3. The method of claim 2 wherein said second DNAsequence further encodes an immunoglobulin hinge region and wherein saidhinge region is joined to said immunoglobulin heavy chain constantregion.
 4. The method of claim 2 wherein said second DNA sequencefurther encodes an immunoglobulin variable region and wherein saidvariable region is joined upstream of and in proper reading frame withsaid immunoglobulin heavy chain constant region domain.
 5. The method ofclaim 2 wherein said host cell is a fungal cell or a cultured mammaliancell.
 6. The method of claim 2 wherein said host cell is a culturedrodent myeloma cell line.
 7. The method of claim 2 wherein saidnon-immunoglobulin polypeptide requiring dimerization for biologicalactivity is selected from the group consisting of a polypeptidecomprising the amino acid sequence of FIG. 1 (Sequence ID Numbers 1 and2) from isoleucine, number 29, to lysine, number 531, a polypeptidecomprising the amino acid sequence of FIG. 1 (sequence ID Numbers 1 and2) from isoleucine, number 29, to methionine, number 441, and apolypeptide comprising the amino acid sequence of FIG. 11 (Sequence IDNumbers 35 and 36) from glutamine, number 24 to glutamic acid, number524.
 8. A method for producing a secreted, biologically active dimerizedpolypeptide fusion, comprising: introducing into a eukaryotic host cella first DNA construct comprising a transcriptional promoter operativelylinked to a first secretory signal sequence followed downstream by andin proper reading frame with a first DNA sequence encoding anon-immunoglobulin polypeptide requiring dimerization for biologicalactivity joined to an immunoglobulin heavy chain constant region domainselected from the group consisting of C_(H)1, C_(H)2, C_(H)3, andC_(H)4; introducing into said host cell a second DNA constructcomprising a transcriptional promoter operatively linked to a secondsecretory signal sequence followed downstream by and in proper readingframe with a second DNA sequence encoding an immunoglobulin light chainconstant region; growing said host cell in an appropriate growth mediumunder physiological conditions to allow the secretion of a biologicallyactive dimerized polypeptide fusion encoded by said first and second DNAsequences; and isolating said biologically active dimerized polypeptidefusion from said host cell.
 9. The method of claim 8 wherein said firstDNA sequence further encodes an immunoglobulin hinge region and whereinsaid hinge region is joined to said immunoglobulin constant region. 10.The method of claim 8 wherein said second DNA sequence further encodesan immunoglobulin variable region and wherein said variable region isjoined upstream of and in proper reading frame with said immunoglobulinlight chain constant region domain.
 11. The method of claim 8 whereinsaid host cell is a fungal cell or a cultured mammalian cell.
 12. Themethod of claim 8 wherein said host cell is a cultured rodent myelomacell line.
 13. The method of claim 8 wherein said non-immunoglobulinpolypeptide requiring dimerization for biological activity is selectedfrom the group consisting of a polypeptide comprising the amino acidsequence of FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number29, to lysine, number 531 a polypeptide comprising the amino acidsequence of FIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number29, to methionine, number 441, and a polypeptide comprising the aminoacid sequence of FIG. 11 (Sequence ID Numbers 35 and 36) from glutamine,number 24 to glutamic acid, number
 524. 14. A method for producing asecreted receptor analog, comprising: introducing into a eukaryotic hostcell a DNA construct comprising a transcriptional promoter operativelylinked to at least one secretory signal sequence followed downstream byand in proper reading frame with a DNA sequence encoding aligand-binding domain of a receptor requiring dimerization forbiological activity joined to a dimerizing protein; growing said hostcell in an appropriate growth medium under physiological conditions toallow the secretion of a receptor analog encoded by said DNA sequence;and isolating said receptor analog from said host cell.
 15. A method fordetermining the presence of PDGF or isoforms thereof in a biologicalsample, comprising: incubating a polypeptide comprising a PDGF receptoranalog fused to a dimerizing protein with a biological sample suspectedof comprising PDGF or an isoform thereof under physiological conditionsto allow the formation of receptor/ligand complexes; and detecting thepresence of the receptor/ligand complexes as an indication of thepresence of human PDGF or an isoform thereof.
 16. The method of claim 15wherein the polypeptide is tagged with a label selected from the groupconsisting of radionuclides, fluorophores, enzymes, and luminescers. 17.The method of claim 15 wherein the biological sample is selected fromthe group consisting of blood, urine, plasma, serum, platelet and othercell lysates, platelet releasates, cell suspensions, cell-conditionedculture media and chemically or physically separated portions thereof.18. The method of claim 15 wherein said human PDGF receptor analog isselected from the group consisting of the amino acid sequence of FIG. 1(sequence ID Numbers 1 and 2) from isoleucine, number 29, to methionine,number 441, joined to a dimerizing protein, the amino acid sequence ofFIG. 1 (Sequence ID Numbers 1 and 2) from isoleucine, number 29, tolysine, number 531, joined to a dimerizing protein and the amino acidsequence of FIG. 11 (Sequence ID Numbers 35 and 36) from glutamine,number 24 to glutamic acid, number 524, joined to a dimerizing protein.19. The method of claim 15 wherein said dimerizing protein comprises atleast a portion of a protein selected from the group consisting of animmunoglobulin light chain, an immunoglobulin heavy chain and yeastinvertase, wherein said portion associates as a dimer in a covalent or anoncovalent manner.
 20. A method for producing a secreted PDGF receptoranalog, comprising: introducing into a eukaryotic host cell a DNAconstruct comprising a transcriptional promoter operatively linked to asecretory signal sequence followed downstream in proper reading frame bya DNA sequence encoding a ligand-binding domain of a PDGF receptor;growing said host cell in an appropriate growth medium underphysiological conditions to allow the secretion of a PDGF receptoranalog encoded by said DNA sequence; and isolating said PDGF receptoranalog from said host cell.
 21. A method for producing a secreted PDGFreceptor analog, comprising: introducing into a cultured rodent myelomacell a DNA construct comprising a transcriptional promoter operativelylinked to a PDGF receptor signal sequence followed downstream by and inproper reading frame with a DNA sequence encoding the amino acidsequence of FIG. 11 (Sequence ID Numbers 35 and 36) from glutamine,number 24, to glutamic acid, number 524, joined to a dimerizing protein,wherein said dimerizing protein is an immunoglobulin constant regionselected from the group consisting of C_(H)1, C_(H)2, C_(H)3, C_(H)4 andC_(κ) joined to an immunoglobulin hinge region; growing said culturedrodent myeloma cell in an appropriate growth medium under physiologicalconditions to allow the secretion of a PDGF receptor analog encoded bysaid DNA sequence; and isolating the PDGF receptor analog from saidcultured rodent myeloma cell.
 22. A method for producing a secreted PDGFreceptor analog, comprising: introducing into a cultured rodent myelomacell a first DNA construct comprising a transcriptional promoteroperatively linked to a PDGF receptor signal sequence followeddownstream by and in proper reading frame with a first DNA sequenceencoding the amino acid sequence of FIG. 1 (Sequence ID Numbers 1 and 2)from isoleucine, number 29, to lysine, number 531 joined to animmunoglobulin light chain constant region; introducing into saidcultured rodent myeloma cell a second DNA construct comprising atranscriptional promoter operatively linked to a PDGF receptor signalsequence followed downstream by and in proper reading frame with asecond DNA sequence encoding the amino acid sequence of FIG. 1 (sequenceID Numbers 1 and 2) from isoleucine number 29, to lysine, number 531joined to an immunoglobulin heavy chain constant region domain selectedfrom the group consisting of C_(H)1, C_(H)2, C_(H)3, and C_(H)4 joinedto an immunoglobulin hinge region; growing said cultured rodent myelomacell in an appropriate growth medium under physiological conditions toallow the secretion of a PDGF receptor analog encoded by said first andsecond DNA sequences; and isolating said PDGF receptor analog from saidcultured rodent myeloma cell.
 23. A method for producing a secreted PDGFreceptor analog, comprising: introducing into a cultured rodent myelomacell a first DNA construct comprising a mouse V_(H) promoter operativelylinked to a PDGF receptor signal sequence followed downstream of and inproper reading frame with a DNA sequence encoding the amino acidsequence of FIG. 11 (Sequence ID Numbers 35 and 36) from glutamine,number 24 to glutamic acid, number 524, joined to an immunoglobulinheavy chain constant region domain selected from the group consisting ofC_(H)1, CH², C_(H)3 and C_(H)4 joined to an immunoglobulin hinge region;introducing into said cultured rodent myeloma cell a second DNAconstruct comprising a mouse V_(κ) promoter operatively linked to a PDGFreceptor signal sequence followed downstream of and in proper readingframe with a DNA sequence encoding the amino acid sequence of FIG. 11(Sequence ID Numbers 35 and 36) from glutamine, number 24 to glutamicacid, number 524, joined to an immunoglobulin light chain constantregion; growing said cultured rodent myeloma cell in an appropriategrowth medium under physiological conditions to allow the secretion of aPDGF receptor analog encoded by said first and second DNA sequences; andisolating said PDGF receptor analog from said cultured myeloma cell. 24.A method for producing a secreted PDGF receptor analog, comprising:introducing into a cultured rodent myeloma cell a first DNA constructcomprising a mouse V_(H) promoter operatively linked to a PDGF receptorsignal sequence followed downstream of and in proper reading frame witha DNA sequence encoding the amino acid sequence of FIG. 1 (Sequence IDNumbers 1 and 2) from isoleucine number 29, to lysine, number 531,joined to an immunoglobulin heavy chain constant region domain selectedfrom the group consisting of C_(H)1, C_(H)2, C_(H)3 and C_(H)4 joined toan immunoglobulin hinge region; introducing into said cultured rodentmyeloma cell a second DNA construct comprising a mouse V_(κ) promoteroperatively linked to a PDGF receptor signal sequence followeddownstream of and in proper reading frame with a DNA sequence encodingthe amino acid sequence of FIG. 11 (Sequence ID Numbers 35 and 36) fromglutamine, number 24 to glutamic acid, number 524, joined to animmunoglobulin light chain constant region; growing said cultured rodentmyeloma cell in an appropriate growth medium under physiologicalconditions to allow the secretion of a PDGF receptor analog encoded bysaid first and second DNA sequences; and isolating said PDGF receptoranalog from said cultured myeloma cell.
 25. A method for producing asecreted PDGF receptor analog, comprising: introducing into a culturedrodent myeloma cell a first DNA construct comprising a mouse V_(H)promoter operatively linked to a PDGF receptor signal sequence followeddownstream of and in proper reading frame with a DNA sequence encodingthe amino acid sequence of FIG. 11 (Sequence ID Numbers 35 and 36) fromglutamine, number 24 to glutamic acid, number 524, joined to animmunoglobulin heavy chain constant region domain selected from thegroup consisting of C_(H)1, C_(H)2, C_(H)3 and C_(H)4 joined to animmunoglobulin hinge region; introducing into said cultured rodentmyeloma cell a second DNA construct comprising a mouse V_(κ) promoteroperatively linked to a PDGF receptor signal sequence followeddownstream of and in proper reading frame with a DNA sequence encodingthe amino acid sequence of FIG. 1 (Sequence ID Numbers 1 and 2) fromisoleucine number 29, to lysine, number 531, joined to an immunoglobulinlight chain constant region; growing said cultured rodent myeloma cellin an appropriate growth medium under physiological conditions to allowthe secretion of a PDGF receptor analog encoded by said first and secondDNA sequences; and isolating said PDGF receptor analog from saidcultured myeloma cell.
 26. A method for determining the presence of PDGFor an isoform thereof in a biological sample comprising the steps of:incubating a polypeptide comprising a PDGF receptor analog joined to adimerizing protein with a biological sample suspected of containing PDGFor an isoform thereof under conditions that allow the formation ofreceptor/ligand complexes; and detecting the presence of receptor/ligandcomplexes, and therefrom determining the presence of human PDGF or anisoform thereof.
 27. The method according to claim 26 wherein saidbiological sample is selected from the group consisting of blood, urine,plasma, serum, platelet and other cell lysates, platelet releasates,cell suspensions, cell-conditioned culture media, and chemically orphysically separated portions thereof.
 28. A method for purifying PDGFor an isoform thereof from a sample, comprising: immobilizing apolypeptide comprising a PDGF receptor analog fused to a dimerizingprotein on a substrate; contacting a sample comprising PDGF or anisoform thereof with the immobilized polypeptide under conditions suchthat the PDGF or isoform thereof binds to the polypeptide; and elutingthe PDGF or isoform thereof from the polypeptide.